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Primer pairs and kit for identifying mating type of Coriolopsis trogii protoplast monokaryon and application of primer pairs

A technology of P. pilosa and protoplasts, which is applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc., can solve the problems of great influence on accuracy, long time consumption, low efficiency, etc. , to achieve accurate and rapid identification, high accuracy and reliability

Active Publication Date: 2020-12-25
广东省科学院生物与医学工程研究所
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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 Coriolopsis trogii protoplast monokaryon and application of primer pairs
  • Primer pairs and kit for identifying mating type of Coriolopsis trogii protoplast monokaryon and application of primer pairs
  • Primer pairs and kit for identifying mating type of Coriolopsis trogii protoplast monokaryon and application of primer pairs

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0059] 1. Preparation of Monokaryon from Protoplasts of P.

[0060] Reagent:

[0061] The culture medium of Giacoma ciliates includes PDA medium, PDB medium and regeneration medium.

[0062] The preparation method of PDA medium is as follows: 15.6g Difco TM Potato Dextrose Agar (Becton, Dickinson and Company, Sparks, MD, USA) was dissolved in 400mL ddH 2 In O, sterilize at 121°C for 15min.

[0063] PDB medium: 2.4g Difco TM Potato Dextrose Broth (Becton, Dickinson and Company, Sparks, MD, USA) was dissolved in 100 mL ddH 2 In O, sterilize at 121°C for 15min.

[0064] The preparation method of regeneration medium is: 15.6g Difco TM Potato Dextrose Agar with 82.15g sucrose dissolved in 400mL ddH 2 In O, sterilize at 121°C for 15min.

[0065] Preparation and Regeneration of Protoplasts from P. gracilis:

[0066] 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 2 days; , Enzymolysis at ...

Embodiment 1

[0073] Embodiment 1: The pair of primers that is used for distinguishing the mating type of the protoplast monokaryon of Pyradocomyces gracilis:

[0074] A pair of primers for identifying the mating type of the protoplast monokaryon of Piacrotomus grisa, the nucleic acid sequence of the primer pair is as follows:

[0075] PMD_1F: 5'-GGCAGTTGTTGTTCTCAGTGT-3' (SEQ ID NO.1);

[0076] PMD_1R: 5'-GGATCATCAGCAATGGTCAGTT-3' (SEQ ID NO.2);

[0077] The specific primer pair was designed according to the whole genome sequence of Piacrotomus clastica (strain number S0301).

[0078] Use the rapid DNA extraction detection kit KG203 (Tiangen Biochemical Technology (Beijing) Co., Ltd.) to extract the genomic DNA of the protoplast monokaryotic strain of Giacoma gracilis, including the following steps:

[0079] 1. Cultivate each monokaryon strain on a PDA plate at 28°C for 3 days;

[0080] 2. Pick mycelium into a 1.5mL centrifuge tube, add 100μL buffer B1;

[0081] 3. Use a grinding pestle...

Embodiment 2

[0086] Embodiment 2: The pair of primers that are used for distinguishing the mating type of the protoplast monokaryon of Piacrotomus ciliformis:

[0087] A pair of primers for identifying the mating type of the protoplast monokaryon of Piacrotomus grisa, the nucleic acid sequence of the primer pair is as follows:

[0088] PMD_2F: 5'-TTCGGTGTGCCTCTCCTATG-3' (SEQ ID NO.3);

[0089] PMD_2R: 5'-CGTTGCGTAGTAGTAGTTGATGA-3' (SEQ ID NO. 4).

[0090] The specific primer pair was designed according to the whole genome sequence of Piacrotomus clastica (strain number S0301).

[0091] Use the rapid DNA extraction detection kit KG203 (Tiangen Biochemical Technology (Beijing) Co., Ltd.) to extract the genomic DNA of the protoplast monokaryotic strain of Giacoma gracilis, including the following steps:

[0092] 1. Cultivate each monokaryon strain on a PDA plate at 28°C for 3 days;

[0093] 2. Pick mycelium into a 1.5mL centrifuge tube, add 100μL buffer B1;

[0094] 3. Use a grinding pest...

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Abstract

The invention discloses primer pairs and kit for identifying the mating type of Coriolopsis trogii protoplast monokaryon and an application of the primer pairs. The invention discloses two primer pairs PMD_1 and PMD_2 for identifying the mating type of the Coriolopsis trogii protoplast monokaryon. According to the invention, genomic DNA of the to-be-identified Coriolopsis trogii protoplast monokaryon is used as a template, the primers PMD_1 and PMD_2 are used for amplification, and the mating type of the Coriolopsis trogii 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, has high accuracy, and can be suitable for research in the related fields of genetic breeding, genomics and the like of edible and medicinal fungi, especially Coriolopsis trogii.

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 test kit and an application thereof for identifying the mating type of the protoplast monokaryon of the sclerocystic bacteria. Background technique [0002] Coriolopsis trogii belongs to Basidiomycota and Polyporaceae. It is a wild fungus resource with large reserves and can grow on living poplar and other trees. Has a strong ability to decompose lignocellulose. Piacrotomus pilus and its related species are mainly used as a laccase-producing fungus in scientific research and production, and have strong pharmacological effects, which can exert anti-cancer effects. In addition, the hyphae and fruiting bodies of G. pilaris have strong ability to withstand high temperature, and the growth and development cycle is relatively short. The above advantages make it a good material for studying the development of polypo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12N15/11
CPCC12Q1/6895C12Q2600/156
Inventor 王丽宁卢传礼马年方
Owner 广东省科学院生物与医学工程研究所
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