CN111926107A - Molecular marker for identifying Pleurotus citrinopileatus and application thereof - Google Patents

Abstract

The invention relates to the technical field of genetic engineering, in particular to a molecular marker for identifying Pleurotus citrinopileatus and application thereof. The primer pair can be used for specifically designing a molecular marker, a probe and/or a kit aiming at the white fungus to screen and identify the white fungus, the white fungus strain can be simply, conveniently, quickly and accurately screened by using the method, and the method has important significance for genetic research and market popularization of the white fungus strain.

Description

Molecular marker for identifying Pleurotus citrinopileatus and application thereof

[ technical field ] A method for producing a semiconductor device

The invention relates to the technical field of genetic engineering, in particular to a molecular marker for identifying Pleurotus ostreatus and application thereof.

[ background of the invention ]

The black fungus (Auricularia heimuer) is a common name of black fungus in southern China, and belongs to the genus Auricularia in the family of Auriculariales, the class of Heterobasidiomycota. Black fungus is widely distributed in China, main production areas comprise Jilin, Heilongjiang, Liaoning, inner Mongolia, Guangxi, Yunnan, Guizhou, Sichuan, Hubei, Shaanxi, Zhejiang and other provinces, the yield is second to that of mushroom and oyster mushroom, and the black fungus is one of four edible fungus cultivation varieties in China.

The Baichou black fungus refers to wild black fungus naturally distributed in counties (regions) such as Tianlin county, Tianyang, Youjiang, Lingyun, Haoye, Longlin, Xilin and Tiandong in Baichou region of Guangxi, and belongs to typical ecological populations suitable for geographical conditions of tropical vegetation in south Asia. The Baicai black fungus is a unique precious black fungus variety in Guangxi, has the excellent qualities of high temperature resistance, high fruiting body soaking rate, bright color, soft texture, tender and smooth mouth feel, excellent mouthfeel and the like, and has once obtained the first-class prize of foreign trade specialties in China.

The white fungus strain and the black fungus commercial strain outside Guangxi have obvious difference: firstly, in the characteristics of hypha culture and the appearance of the fruit body, the pigment generation is early during the hypha culture of most strains, the color of the fruit body is lighter and semitransparent, the texture of the fruit body is soft and tender, and the chewiness is low, and the pigment generation is late or no pigment during the hypha culture, the color of the fruit body is dark and opaque, the texture of the fruit body is crisp, and the chewiness is high; secondly, in the genetic background, the genetic relationship between the two is far away, and according to the ISSR cluster analysis result, the two belong to different ecological groups.

Although there are many differences between the Pleurotus ostreatus strain and the foreign commercial strain of Auricularia auricula in Guangxi, the appearance of fruiting bodies is greatly influenced by environmental conditions, and the fruiting bodies lack quantitative indexes, so that errors in identification of the Pleurotus ostreatus strain and the commercial strain of Auricularia auricula in Guangxi are easily caused in actual production and scientific research application; although the ISSR analysis technology can distinguish two types of black fungus varieties, the technology also has the defects of unstable banding patterns, uncertain characteristic banding sequences and the like, and often needs a plurality of primers, the fingerprint of the ISSR is also complex, the software analysis is needed, and the process is complicated. Therefore, there is a need to develop a molecular marker which can simply, rapidly and clearly identify the Pleurotus ostreatus strain and the exotic commercial strain of Auricularia, and can rapidly distinguish the Pleurotus ostreatus.

[ summary of the invention ]

In view of the above, there is a need to develop a molecular marker that can simply, rapidly and clearly identify the strain of Pleurotus ostreatus and the commercial strain of Auricularia auricular, and can rapidly distinguish Pleurotus ostreatus.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

pair of molecular marker primers, method for producing the same, and kit for producing the same

The sequence of the upstream primer is as follows: 5'-CAGATATGCAGCACATCACAC-3', respectively;

the sequence of the downstream primer is as follows: 5'-CCAACCAAACCAGAAACATCC-3' are provided.

The invention also comprises a probe and/or a kit of the molecular marker primer pair.

The invention also comprises the application of the molecular marker primer pair and the probe and/or the kit in identifying the white fungus.

The invention also comprises a method for identifying the white fungus by using the molecular marker primer pair and/or the probe and/or the kit, wherein the method comprises the following steps: extracting DNA of different samples of the black fungus, carrying out PCR reaction with the primer pair, the probe or the kit, carrying out gel electrophoresis detection, if 635bp can be amplified, the germplasm source of the black fungus is proved to be from Guangxi Baicai, and if 635bp cannot be amplified, the germplasm source of the black fungus is proved not to be from Guangxi Baicai.

Further, the reaction system of the PCR reaction is as follows: 2 × Es TaqMasterMix 11.5 μ L, ddH₂10.5 mul of O, 1 mul of 10 mul mol/L upstream primer, 1 mul of 10 mul mol/L downstream primer and 1 mul of genome DNA of black fungus strain to be detected

The reaction procedure for PCR amplification was: pre-denaturation at 95 deg.C for 5min, denaturation at 95 deg.C for 30sec, annealing at 60 deg.C for 30sec, extension at 72 deg.C for 60sec, circulation for 30 times, extension at 72 deg.C for 7min, and storage at 4 deg.C for 30 min.

The invention also comprises a specific fragment amplified from the white fungus by the molecular marker primer pair, and the nucleic acid sequence of the fragment is shown as the sequence table SEQ ID NO. 3.

The invention has the following beneficial effects:

1. the primer pair has strong specificity, can specifically amplify the Pleurotus ostreatus DNA from a hundred of colors, can amplify a 635bp fragment, and has no amplification effect on other Pleurotus ostreatus strains. The primer pair can be used for specifically designing a molecular marker, a probe and/or a kit aiming at the white fungus to screen and identify the white fungus, the white fungus strain can be simply, conveniently, quickly and accurately screened by using the method, and the method has important significance for genetic research and market popularization of the white fungus strain.

[ description of the drawings ]

FIG. 1 is a fingerprint of common features of ISSR products of Pleurotus ostreatus strain recovered from glue of example 1; in the figure, M is Maker DL2000 which is 2000bp, 1000bp, 750bp, 500bp, 250bp and 100bp from top to bottom in sequence; numbers 1 to 4: from left to right, Guiyun No. 3, ty013, ly041 and ly051 are arranged in sequence.

FIG. 2 is an electrophoretogram of the amplification of the pair of primers of example 2 against a strain of Coprinus comatus from a different germplasm source; in the figure, M is Maker DL2000 which is 2000bp, 1000bp, 750bp, 500bp, 250bp and 100bp from top to bottom in sequence; numbers 1-23: from left to right, sequentially comprises Ty013, Guiyun No. 3, ly031, ly041, ly051, Ty063, Baiyun No. 6, xy186-1, xy185-3, Ty181-5, Ty062, xy182-5, TE011, Coprinus comatus, Xinke, Auricularia auricula No. 2, Auricularia auricula 149, Heishi, Xinke No. 10, New Keke No. 916, New Keke No. 4, Au8129 and Black 29.

[ detailed description ] embodiments

All of the features disclosed in this specification, or all of the steps in any method or process so disclosed, may be combined in any combination, except combinations of features and/or steps that are mutually exclusive.

Any feature disclosed in this specification (including any accompanying claims, abstract) is merely an example of a generic series of equivalent or similar features, unless explicitly described as such.

Example 1:

genetic difference analysis is carried out on the Pleurotus citrinopileatus and exotic black fungus commercial strains (samples are selected from Pleurotus citrinopileatus 1: Guiyun No. 3, Ty013, ly041, ly051, Ty063, Ty071, YE1 and Baiyun No. 6; Pleurotus citrinopileatus 916; and Pleurotus citrinopileatus New family No. 4 from Jiangsu Jiangdu), and the result shows that the Pleurotus citrinopileatus with germplasm resources from the Pleurotus citrinopileatus has the same specific band of about 1000bp, the band is subjected to gel cutting and recovery, and the fingerprint band of the recovered gel is shown in figure 1, wherein M is: DL2000, which is 2000bp, 1000bp, 750bp, 500bp, 250bp and 100bp from top to bottom in sequence; numbers 1 to 4: from left to right, Guiyun No. 3, Ty013, ly041 and ly051 (all of which are all Baise black fungus) are sequentially arranged, as can be seen from figure 1, samples 1-4 have an obvious public strip at about 1000bp, and the public strip is subjected to gel cutting and recovery; sequencing by taking a DNA fragment of the Pleurotus ostreatus strain ty063 as a template, wherein the specific sequence is shown as a sequence table SEQ ID NO. 4; the primers are designed according to the sequence table SEQ ID NO. 4 as follows:

3-TY063-5F: 5'-CAGATATGCAGCACATCACAC-3'; (shown in SEQ ID NO:1 of the sequence Listing)

3-TY063-5R: 5'-CCAACCAAACCAGAAACATCC-3' (shown in SEQ ID NO:2 of the sequence Listing).

Example 2:

multiple samples were amplified using the primer sequences of example 1: the sources of the amplified samples of this example are shown in table 1:

TABLE 1 species name and source of different strains of Auricularia

Respectively extracting DNA of the No. 1-23 samples, and amplifying by adopting a PCR amplification method, wherein the reaction system of the PCR is as follows: 2 × Es TaqMasterMix 11.5 μ L, ddH₂10.5 mul of O, 1 mul of 10 mul mol/L upstream primer, 1 mul of 10 mul mol/L downstream primer and 1 mul of genome DNA of the black fungus strain to be detected;

the reaction procedure for PCR amplification was: pre-denaturation at 95 deg.C for 5min, denaturation at 95 deg.C for 30sec, annealing at 60 deg.C for 30sec, extension at 72 deg.C for 60sec, circulation for 30 times, extension at 72 deg.C for 7min, and storage at 4 deg.C for 30 min.

The reaction results are shown in FIG. 2: m: DL2000 (2000 bp, 1000bp, 750bp, 500bp, 250bp and 100bp from top to bottom), lanes 1-12 are Pleurotus ostreatus strains: ty013, Guiyun No. 3, ly031, ly041, ly051, ty063, Baiyun No. 6, xy186-1, xy185-3, ty181-5, ty062, xy 182-5; lanes 13-14 are Auricularia brachycarpa strain TE011 and Coprinus comatus; 15-23 are respectively commercial black fungus strains: new family, black fungus No. 2, black fungus 149, black fungus No. three, new family No. 10, 916, new family No. 4, Au8129, black 29. As can be seen, all of the Pleurotus ostreatus strains (lanes 1-12) have a characteristic fingerprint band with a size of 500-750bp, while the non-Pleurotus ostreatus strains (lanes 13-23) cannot amplify the characteristic fingerprint band, indicating that the characteristic fingerprint band is unique to the Pleurotus ostreatus strains. Therefore, the specific primer can be used for distinguishing the white black fungus strain from other commercial black fungus strains; the public fragment is cut with glue, recovered and sequenced to obtain a gene sequence with the size of 635bp, and the sequence is shown as a sequence table SEQ ID NO. 3.

Example 3:

the person skilled in the art can design kits or probes containing the above primer pairs for the specific identification of the Pleurotus ostreatus based on the primer pairs of examples 1 and 2, and therefore it is another object of the present invention to protect a kit comprising the above primer pair 3-TY063-5F: 5'-CAGATATGCAGCACATCACAC-3'; 3-TY063-5R: 5'-CCAACCAAACCAGAAACATCC-3'; if the molecular marker, the kit or the probe can amplify 635bp in the black fungus DNA, the black fungus variety is proved to be from a hundred-color black fungus, and if the size cannot be amplified, the black fungus variety is not from a hundred-color black fungus.

In conclusion, the primers can well distinguish the Pleurotus ostreatus from the Pleurotus ostreatus of other limbus by amplifying the common fragments of the Pleurotus ostreatus, can quickly identify the Pleurotus ostreatus, and can improve the identification efficiency and accuracy from the perspective of molecular biology.

The above examples are merely illustrative of several embodiments of the present invention, and the description thereof is more specific and detailed, but not to be construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present invention should be subject to the appended claims.

Sequence listing

<110> institute of microbiology of Guangxi Zhuang autonomous region academy of agricultural sciences

<120> molecular marker for identifying Pleurotus ostreatus and application thereof

<141> 2020-09-15

<160> 4

<170> SIPOSequenceListing 1.0

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<213> Black fungus (Auricularia auricula-judae)

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cagatatgca gcacatcaca c 21

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<213> Black fungus (Auricularia auricula-judae)

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ccaaccaaac cagaaacatc c 21

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<213> Black fungus (Auricularia auricula-judae)

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cagatatgca gcacatcaca cctcaggcat tgccctgcat tgccggtgcg cgctgggagt 60

acaggcaaag ctgcaacgat cttgccgatg tcaatccttc accctctgtc ccatggatgc 120

tcgaccgctc cagtacctcg gacctcggcc gccacactct cctcggtccg cgctcaacac 180

tgtccatggt ccggaggatg tctggacaca ttgggagtct tgattgttgg gtggtgcgct 240

tgtaccgcgc ttgctgacgt gttgcactca ctgctgcgct tgccattgcg tcgcccccct 300

ttctcccatg tcggtgtgtg catacatgca aggacacctc ggtctgtctc agatacccac 360

ccaataccct ccataggcac gggcatcctt tatcaccttt gtccgggtcc ggagtcgttc 420

tgcgctttct gttgcggtct tcgtcgggtc cctcctccac cgcggtttca tttcgccgtc 480

cagcgatcct cgcactcttc ttgggcaccc aagctccttg cgcccggccc cgtggcccct 540

tcgtcagtgt atcctaacgg tctctccgct cagcgagtac ctcggtctct tcccaggaca 600

agggtcatat gacgggatgt ttctggtttg gttgg 635

<210> 4

<211> 878

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<213> Black fungus (Auricularia auricula-judae)

<400> 4

ttcgcgtgtc gcccttagag agagagagag agggacaagg aagaacagac aaggggtaca 60

attgaccaga ctgttatggt gcacggcaat tgctcccata cgctgcttcc gttggatctc 120

ttttgccgct tcgccagctc cgtctgaccg atgtacccgt cccgcaagcc tctccagacc 180

gctggcacct cggatacctt ctctcggcgt caccagaccc ttgtcaacct cggtccttgg 240

atgtgacgca ccattgccat tgtcgttgac cactgctgcg ttggttttca accagcatgt 300

agccgcttgt cggtccgtct ccttggtgtc acattacggc ctacaccttc agatatgcag 360

cacatcacac cttgggcatt gccctgcatt gccggtgcgc gcggggagta caggcaaagc 420

tgcaacgatc ttacttgttg cggtcttcgt cgggtccctc ctcccccgct gtttcatttc 480

gccgtcccgt ggccctcgca cctcggccgg gcgcccaaac tccttgcgcc cggccccgcg 540

gcccttttgt cggcgcgtcc cagcggtctc tccgctcggc gagtacctcg gtctcttccc 600

aggagaaggg taatacgacg ggatgtttct ggtttggttg ggctctattt atcccaggtt 660

tcctgactcg ccgacgacgc ccaggtggtc tctttcggcc tgatctgtgg cgcccgcgtg 720

gcaaccggtc ggtggcgcaa ttggtccgcg ggtcccctcg gccttgcttc cgcagtatat 780

atatctgggc cgtcgcccct cttgtacctt aggtttggtc tggtctatca taccgcttat 840

ccgcgcctct ctctctctct ctaagggcga cacgcgaa 878

Claims (6)

1. The pair of molecular marker primers, characterized in that

The sequence of the upstream primer is as follows: 5'-CAGATATGCAGCACATCACAC-3', respectively;

the sequence of the downstream primer is as follows: 5'-CCAACCAAACCAGAAACATCC-3' are provided.

2. A probe and/or kit comprising a pair of molecularly imprinted primers according to claim 1.

3. Use of the pair of molecular marker primers according to claim 1 and the probe and/or kit according to claim 2 for identifying Pleurotus ostreatus.

4. The method for identifying the white fungus by using the molecular marker primer pair as claimed in claim 1 and/or the probe and/or the kit as claimed in claim 2, is characterized in that the method comprises the following steps: extracting DNA of different samples of the black fungus, carrying out PCR reaction with the primer pair, the probe or the kit, carrying out gel electrophoresis detection, if 635bp can be amplified, the germplasm source of the black fungus is proved to be from Guangxi Baicai, and if 635bp cannot be amplified, the germplasm source of the black fungus is proved not to be from Guangxi Baicai.

5. The method for identifying the white fungus according to claim 4, wherein the reaction system of the PCR reaction is as follows: 2 × Es TaqMasterMix 11.5 μ L, ddH₂10.5 mul of O, 1 mul of 10 mul mol/L upstream primer, 1 mul of 10 mul mol/L downstream primer and 1 mul of genome DNA of black fungus strain to be detected

The reaction procedure for PCR amplification was: pre-denaturation at 95 deg.C for 5min, denaturation at 95 deg.C for 30sec, annealing at 60 deg.C for 30sec, extension at 72 deg.C for 60sec, circulation for 30 times, extension at 72 deg.C for 7min, and storage at 4 deg.C for 30 min.

6. The nucleic acid sequence of the specific fragment amplified from the Pleurotus ostreatus of the molecular marker primer pair according to claim 1 is shown in SEQ ID NO. 3 of the sequence table.

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