Method for identifying lepista sordida protoplast monokaryon mating types and special primer pair SR-6*14 therefor
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A technique for protoplasts and protoplasts of Fructus versicolor is applied in the field of identifying the mating type of protoplasts of Fructus Fructus
Inactive Publication Date: 2013-08-28
BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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preparation example Construction
[0031] 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.
[0032] 1.2 Protoplast preparation
[0033] 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.
[0034] 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.
[0035] 3) Centrifuge the filtrate of step 2) at 3000 rpm for 10 min and collect the precipita...
Embodiment 1
[0049] Example 1. Using the PCR primer pair SR-6×14 to identify the mating type of the protoplast monokaryon of the mushroom
[0050] 1. PCR reagents for identifying or assisting in identifying the mating type of monokaryon protoplasts
[0051] The reagents for identifying or assisting in identifying the mating type of the protoplast monokaryon of Amanita chinensis in this embodiment consist of PCR primer pair SR-6×14, 10×Taq buffer, dNTP mix, Taq DNA polymerase and ddH 2 O composition.
[0052] Among them, the PCR primer pair SR-6×14 is composed of two single-stranded DNAs, SR-6×14-F and SR-6×14-R, and its sequence is as follows:
[0053] SR-6×14-F:5'-TCCAAACCGGTAGGTAACAG-3' (SEQ ID No.1),
[0054] SR-6×14-R: 5'-ATCGTCACATTCGGAGTCAA-3' (SEQ ID No. 2).
[0055] 10×Taq buffer, dNTP mix and Taq DNA polymerase were purchased from Beijing Shengxu Baichuan Company (CNS).
[0056] 2. Identify or assist in the identification of monokaryotic mating types
[0057] Inoculate the 30...
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Abstract
The invention discloses a method for identifying lepista sordida protoplast monokaryon mating types and a special primer pair SR-6*14 therefor. The method comprises the following steps of: with genomic DNA (deoxyribonucleic acid) of two lepista sordida protoplast monokaryon to be identified as templates, carrying out PCR (polymerase chain reaction) amplification by using a PCR primer pair consisting of two single-stranded DNA shown by SEQ ID NO.1 and SEQ ID No.2; and detecting the sizes of the obtained PRC products, wherein if the PCR products of the two lepista sordida protoplast monokaryon to be identified contain or do not contain the DNA fragment of 500bp-800bp, the mating types of the two lepista sordida protoplast monokaryon to be identified are same, if the PCR product of one of the two lepista sordida protoplast monokaryon to be identified contains the DNA fragment of 500bp-800bp, and the PRC product of the other of the two lepista sordida protoplast monokaryon to be identified does not contain the DNA fragment of 500bp-800bp, the mating types of the two lepista sordida protoplast monokaryon to be identified 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-6×14 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...
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IPC IPC(8): C12Q1/68C12N15/11A01H1/02
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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