Method for identifying mating type of protoplasted monokaryon of lepista sordida and special primer pair SR-6*4 thereof

A protoplast, monokaryotic technology, applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc., to achieve high accuracy and reliability

Inactive Publication Date: 2015-03-11
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 mating type of protoplasted monokaryon of lepista sordida and special primer pair SR-6*4 thereof
  • Method for identifying mating type of protoplasted monokaryon of lepista sordida and special primer pair SR-6*4 thereof
  • Method for identifying mating type of protoplasted monokaryon of lepista sordida and special primer pair SR-6*4 thereof

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Experimental program
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Effect test

preparation example Construction

[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 precipitate.

[0037] 4) The precipitate in step 3) was washed three times with MM buffer, and then suspended in 100 μl of MM buffer, which was the protoplast solution.

[0038] 1.3 Ob...

Embodiment 1

[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 mating type of the protoplast monokaryon of A. variegata in this embodiment consist of PCR primer pair SR-6×4, 10×Taq buffer, dNTP mix, Taq DNA polymerase and ddH 2 O composition.

[0053] Among them, the PCR primer pair SR-6×4 is composed of two single-stranded DNAs, SR-6×4-F and SR-6×4-R, and its sequence is as follows:

[0054] SR-6×4-F:5'-AACCGGTAGGCAGTTACTTT-3' (SEQ ID No.1),

[0055] SR-6×4-R: 5'-AGAGCAAGCCTTTCATACAGT-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 monokaryons of the above 1.3 into the PDA medium respectively, culture at 25°C for 7-10 days, scrape about 0.1g of the mycelia...

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Abstract

The invention discloses a method for identifying a mating type of protoplasted monokaryon of lepista sordida and a special primer pair SR-6*4 thereof. The method comprises the following steps of: by taking the genome DNAs of the protoplasted monokaryons of two plants of lepista sordida as templates, respectively, performing PCR (Polymerase Chain Reaction) amplification by using a PCR primer pair composed of two single-stranded DNAs as shown in SEQ ID No.1 and SEQ ID No.2; detecting the sizes of the PCR products obtained; if the PCR products of the protoplasted monokaryons of the two plants of lepista sordida to be identified both contain DNA segments of 500 bp-800 bp or not, indicating that the mating types of the protoplasted monokaryons of the two plants of lepista sordida to be identified are the same; if one plant contains the DNA segments of 500 bp-800 bp while the other plant contains no DNA segment of 500 bp-800 bp, indicating that the mating types of the protoplasted monokaryons of the two plants of lepista sordida to be identified are different.

Description

technical field [0001] The invention relates to a method for identifying the mating type of the protoplast monokaryon of the flower face mushroom and its special primer pair SR-6×4. 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...

Claims

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Application Information

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