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Primer pair, kit and application thereof for identifying mating type of protoplast monokaryon

A technology of Piacrotomus ciliates and protoplasts, which is applied to biochemical equipment and methods, measurement/testing of microorganisms, DNA/RNA fragments, etc., and can solve problems such as large impact on accuracy, long time consumption, and low efficiency , to achieve the effect of high accuracy and reliability

Active Publication Date: 2022-04-22
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 pair, kit and application thereof for identifying mating type of protoplast monokaryon
  • Primer pair, kit and application thereof for identifying mating type of protoplast monokaryon
  • Primer pair, kit and application thereof for identifying mating type of protoplast monokaryon

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Experimental program
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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 a pair of primers, a kit and an application thereof for identifying the mating type of the protoplast monokaryon of Piacrotomus ciliformis. The invention discloses two primer pairs, PMD_1 and PMD_2, for identifying the mating type of the protoplast monokaryon of the Piacrotomus grisa. In the present invention, the genome DNA of the protoplast monokaryon of the Piacrotomus gracilis to be identified is used as a template, amplified with PMD_1 and PMD_2 primers, and the mating type is determined according to the size of the detected PCR product. The method of the present invention is simple and easy, shortens the identification time and simplifies the identification steps, has high accuracy, and is applicable to the research of edible and medicinal fungi, especially the genetic breeding of Piacrotomus pilaris, genomics and other related fields.

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 Patents(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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