Method for identifying protoplast monokaryon mating types of lepista sordida and special primer pair SR-4*2 thereof

A technique for protoplasts and protoplasts of Pleurotus edodes, applied in the field of identifying the mating type of protoplasts of Pleurotus edodes, can solve the problems of narrow temperature range, low yield, poor insect resistance, etc., and achieve high accuracy and reliability Effect

Inactive Publication Date: 2014-11-05
BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in actual production, there are problems such as low yield, poor insect resistance, narrow temperature range, fragile fruiting bodies, and difficult transportation and storage.

Method used

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  • Method for identifying protoplast monokaryon mating types of lepista sordida and special primer pair SR-4*2 thereof
  • Method for identifying protoplast monokaryon mating types of lepista sordida and special primer pair SR-4*2 thereof
  • Method for identifying protoplast monokaryon mating types of lepista sordida and special primer pair SR-4*2 thereof

Examples

Experimental program
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Effect test

preparation example Construction

[0032] The preparation method of the solid regeneration plate (RM): add sorbitol and agar to the liquid MCM medium so that the concentrations are 1M and 20g / L, respectively.

[0033] 1.2 Protoplast preparation

[0034] 1) Take the hyphae of A. japonica and culture them in liquid MCM medium at 160rpm and 25°C for 4 days (3-5 days are acceptable), filter and collect the mycelium with 3 layers of sterile lens paper, and then use 0.6M manna Alcohol aqueous solution washed 2-3 times.

[0035] 2) Suspend the hyphae (about 1 g) of A. japonica in step 1) in 1.5% lysozyme solution (dissolve 1.5 g lysozyme with 0.6M mannitol aqueous solution and set the volume to 100 mL; lysozyme was purchased from Guangdong Bide Biotechnology Co., Ltd., catalog number: Bd_8110001023), in a water-bath shaker (32°C, 60rpm) for 2 hours, filtered with 3 layers of sterile lens tissue and collected the filtrate.

[0036] 3) Centrifuge the filtrate of step 2) at 3000 rpm for 10 min and collect the precipita...

Embodiment 1

[0050] Example 1. Use of PCR primer pair SR-4×2 to identify the mating type of the protoplast monokaryon of the mushroom

[0051] 1. PCR reagents for identifying or assisting in identifying the mating type of monokaryon protoplasts

[0052] The reagents for identifying or assisting in identifying the monokaryon mating type of the protoplasts of A. variegata in this embodiment consist of PCR primer pair SR-4×2, 10×Taq buffer, dNTP mix, Taq DNA polymerase and ddH 2 O composition.

[0053] Among them, the PCR primer pair SR-4×2 is composed of two single-stranded DNAs, SR-4×2-F and SR-4×2-R, and its sequence is as follows:

[0054] SR-4×2-F:5'-CGGCACCTTCCATCATTTAC-3' (SEQ ID No.1),

[0055] SR-4×2-R:5'-TATGATTCCCTCTCCGTCCGT-3' (SEQ ID No. 2).

[0056] 10×Taq buffer, dNTP mix and Taq DNA polymerase were purchased from Beijing Shengxu Baichuan Company (CNS).

[0057] 2. Identify or assist in the identification of monokaryotic mating types

[0058] Inoculate the 30 protoplast mo...

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Abstract

The invention discloses a method for identifying the protoplast monokaryon mating types of lepista sordida and a special primer pair SR-4*2 thereof. The method comprises the following steps of: with genome DNAs (deoxyribonucleic acids) of the protoplast monokaryons of two plants of lepista sordida to be identified as templates, performing PCR (Polymerase Chain Reaction) amplification by using a PCR primer pair composed of two single chain DNAs as shown in SEQ ID No.1 and SEQ ID No.2, and detecting the sizes of the obtained PCR products; if the PCR products of the protoplast monokaryons of the two plants of lepista sordida to be identified both contain or do not obtain 500 bp-800 bp of DNA segments, indicating that the mating types of the protoplast monokaryons of the two plants of lepista sordida are the same; and if one plant contains 500 bp-800 bp of DNA segments, while the other plant contains no 500 bp-800 bp of DNA segments, indicating that the mating types of the protoplast monokaryons of the two plants of lepista sordida are different.

Description

technical field [0001] The invention relates to a method for identifying the monokaryon mating type of the protoplast and the special primer pair SR-4×2 of the mushroom. Background technique [0002] The nutrient incompatibility system is a genetic system for identifying aliens. In most fungi, this system can cause genetically distinct individuals to produce characteristic somatic incompatibility in the junction zone (Qi Yuancheng et al., Chinese cultivated pellagra Comparison of somatic cell incompatibility test and RAPD analysis results of Pleurotus species. Acta Mycophyta Sinica, 2010, 29 (3)) reaction, the intensity of somatic cell incompatibility reaction varies from individual to individual. In fungi, nutritional incompatibility is also known as somatic incompatibility or heterokaryotic incompatibility. fusion to maintain individual genetic stability. The mating type is the type of combination determined according to whether the individuals of the mating factors can ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/11
Inventor 许峰刘宇赵爽王守现李登进王鹏耿小丽王兰青
Owner BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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