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Primer pairs and kit for identifying mating type of ganoderma lucidum protoplast monokaryon and application of primer pairs

A technology of protoplasts and primer pairs, applied in the direction of microorganism-based methods, microorganisms, biochemical equipment and methods, etc., can solve the problems of large influence on accuracy, long time-consuming, low efficiency, etc., and achieve high accuracy and reliability , accurate and rapid identification of the effects of

Pending Publication Date: 2020-12-25
GUANGDONG ACAD OF SCI INST OF BIOENGINEERING
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method takes a long time, has low efficiency, and the accuracy is greatly affected by the experimenter's subjective

Method used

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  • Primer pairs and kit for identifying mating type of ganoderma lucidum protoplast monokaryon and application of primer pairs
  • Primer pairs and kit for identifying mating type of ganoderma lucidum protoplast monokaryon and application of primer pairs
  • Primer pairs and kit for identifying mating type of ganoderma lucidum protoplast monokaryon and application of primer pairs

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0063] 1. Preparation of Ganoderma lucidum protoplast monokaryon

[0064] Reagent:

[0065] Ganoderma lucidum medium, including PDA medium, PDB medium and regeneration medium.

[0066] PDA medium: 15.6g Difco TM Potato Dextrose Agar (Becton, Dickinson and Company, Sparks, MD, USA) was dissolved in 400mL ddHO 2 In O, sterilize at 121°C for 15min.

[0067] PDB medium: 2.4g DifcoTM Potato Dextrose Broth (Becton, Dickinson and Company, Sparks, MD, USA) was dissolved in 100mL ddH 2 In O, sterilize at 121°C for 15min.

[0068] Regeneration Medium: 15.6g Difco TM Potato Dextrose Agar with 82.15g sucrose dissolved in 400mLddH 2 In O, sterilize at 121°C for 15min.

[0069] Ganoderma lucidum protoplast preparation and regeneration:

[0070] Use a homogenizer to crush 1 g of the mycelia cultured on the PDA plate for 5 days, divide into PDB medium, and culture at 28°C for 3 days; Enzymolysis at 60rpm / min for 2h; filter with sterile non-defatted cotton to remove residual hyphae ...

Embodiment 1

[0077] Example 1: Primer pairs for identifying the mating type of Ganoderma lucidum protoplast monokaryon:

[0078] The primer pair for identifying the mating type of Ganoderma lucidum protoplast monokaryon, the nucleic acid sequence of the primer pair is as follows:

[0079] Glpm_1F: 5'-ATCTCCTCCATCGCCTGTTC-3' (SEQ ID NO.1);

[0080] Glpm_1R: 5'-TCTTGTGACGCTCTGTTCCT-3' (SEQ ID NO.2);

[0081] The specific primer pair is based on two different mating types GL012_8 (mating type is A y B y ) and GL012_53 (mating type is A x B x ) Whole-genome sequence design of a monokaryon.

[0082] Use rapid DNA extraction detection kit KG203 (Tiangen Biochemical Technology (Beijing) Co., Ltd.) to extract Ganoderma lucidum protoplast monokaryon strain genomic DNA, including the following steps:

[0083] 1. Cultivate each monokaryon strain on a PDA plate at 28°C for 5 days;

[0084] 2. Pick mycelium into a 1.5mL centrifuge tube, add 100μL buffer B1;

[0085] 3. Use a grinding pestle to ...

Embodiment 2

[0090] Example 2: Primer pairs for identifying the mating type of Ganoderma lucidum protoplast monokaryon:

[0091] The primer pair for identifying the mating type of Ganoderma lucidum protoplast monokaryon, the nucleic acid sequence of the primer pair is as follows:

[0092] Glpm_2F: 5'-TCGTCCTCACTTCCATCCATC-3' (SEQ ID NO.3);

[0093] Glpm_2R: 5'-GGTTGTTGCTCGGTCTCATAC-3' (SEQ ID NO. 4).

[0094] The specific primer pair was based on two different mating types GL012_8 (mating type A y B y ) and GL012_53 (mating type is A x B x ) the whole genome sequence of the monokaryon, by means of comparative genomics, locate the unique genome sequence fragments of the two strains and design related primers.

[0095] Use rapid DNA extraction detection kit KG203 (Tiangen Biochemical Technology (Beijing) Co., Ltd.) to extract Ganoderma lucidum protoplast monokaryon strain genomic DNA, including the following steps:

[0096] 1. Cultivate each monokaryon strain on a PDA plate at 28°C for 5...

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Abstract

The invention discloses primer pairs and kit for identifying the mating type of ganoderma lucidum protoplast monokaryon and an application of the primer pairs. The invention discloses three groups ofprimer pairs Glpm_1, Glpm_2 and Glpm_3 for identifying the mating type of the ganoderma lucidum protoplast monokaryon. According to the invention, genomic DNA of the ganoderma lucidum protoplast monokaryon to be identified is used as a template, the primers Glpm_1, Glpm_2 and Glpm_3 are used for amplification, and the mating type of the ganoderma lucidum protoplast monokaryon is determined according to the size of a PCR product obtained by detection. The method provided by the invention is simple and easy to implement, shortens the identification time, simplifies the identification steps, hashigh accuracy, and can be suitable for research in the related fields of genetic breeding, genomics and the like of edible and medicinal fungi, especially ganoderma lucidum.

Description

technical field [0001] The invention belongs to the technical field of identification of edible and medicinal fungi, and in particular relates to a pair of primers, a kit and an application thereof for identifying mating types of ganoderma protoplasts and monokaryons. Background technique [0002] Ganoderma lucidum (Ganoderma lucidum) belongs to Basidiomycota (Ganoderma lucidum) in classification, Ganoderma family (Ganodermataceae) Ganoderma (Ganoderma). Ganoderma lucidum is a precious traditional Chinese medicine, which has the functions of strengthening the body and strengthening the body. Modern pharmacological and clinical studies have shown that Ganoderma lucidum has significant effects on anti-tumor, anti-virus, regulating blood sugar, and enhancing immunity. Triterpenes and polysaccharides are the main active ingredients in Ganoderma lucidum. In addition, as one of the representatives of white-rot fungi, Ganoderma lucidum can use its own lignin oxidase system to com...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12Q1/686C12Q1/04C12N15/11C12R1/645
CPCC12Q1/6895C12Q1/686
Inventor 王丽宁黄清铧李锦荣
Owner GUANGDONG ACAD OF SCI INST OF BIOENGINEERING
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