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Method for identification or auxiliary identification of mating type of protoplast monokaryon of Lepista sordid and special primer pair SR-5*16 used therein

A technology of protoplasts and cauliflower, applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc., can solve the problems of narrow adaptation temperature range, poor insect resistance, low yield, etc. high performance and reliability

Active Publication Date: 2013-07-10
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 identification or auxiliary identification of mating type of protoplast monokaryon of Lepista sordid and special primer pair SR-5*16 used therein
  • Method for identification or auxiliary identification of mating type of protoplast monokaryon of Lepista sordid and special primer pair SR-5*16 used therein
  • Method for identification or auxiliary identification of mating type of protoplast monokaryon of Lepista sordid and special primer pair SR-5*16 used therein

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Experimental program
Comparison scheme
Effect test

preparation example Construction

[0029] 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.

[0030] 1.2 Protoplast preparation

[0031] 1) Take the mycelia of Azalea variegata in liquid MCM medium, culture at 160rpm, 25°C for 4 days (3-5 days are acceptable), filter with 3 layers of sterile lens paper to collect the mycelia, and then wash with 0.6M mannitol Wash with aqueous solution 2-3 times.

[0032] 2) Suspend the mycelia (about 1 g) of 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 Bi Germany Biotechnology Co., Ltd., product catalog number: Bd_8110001023), in a water-bath shaker (32 ° C, 60 rpm) for 2 hours, filtered with 3 layers of sterile lens tissue and collected the filtrate.

[0033] 3) Centrifuge the filtrate of step 2) at 3000 rpm for 10 min and collect the precipita...

Embodiment 1

[0046] Example 1. Using PCR primer pair SR-5×16 to identify the mating type of the protoplast monokaryon of Pleurotus chinensis

[0047] 1. PCR reagents for identifying or assisting in identifying the mating type of monokaryon protoplasts

[0048] The reagents for identifying or assisting in identifying the mating type of protoplast monokaryon in this embodiment consist of PCR primer pair SR-5×16, 10×Taq buffer, dNTP mix, Taq DNA polymerase and ddH 2 O composition.

[0049] Among them, the PCR primer pair SR-5×16 is composed of two single-stranded DNAs, SR-5×16-pre-F and SR-5×16-pre-R, and its sequence is as follows:

[0050] SR-5×16-pre-F:5'-ATCAGGTAGGTAGGCGTAAC-3' (SEQ ID No.1),

[0051] SR-5 x 16-pre-R: 5'-TGCGTACGAATTCGGGTAA-3' (SEQ ID No. 2).

[0052] 10×Taq buffer, dNTP mix and Taq DNA polymerase were purchased from Beijing Shengxu Baichuan Company (CNS).

[0053] 2. Identify or assist in the identification of monokaryotic mating types of protoplasts

[0054] Inocul...

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Abstract

The invention discloses a method for identification or auxiliary identification of the mating type of the protoplast monokaryon of Lepista sordid and a special primer pair SR-5*16 used therein. The method comprises the following steps: with genome DNAs of the protoplast monokaryon of two to-be-identified Lepista sordid strains as templates respectively, carrying out PCR amplification by using a PCR primer pair as represented by SEQ ID No. 1 and 2; and detecting the size of obtained PCR products, determining that the mating types of the protoplast monokaryon of the two to-be-identified Lepista sordid strains are identical if both PCR products of the protoplast monokaryon of the two Lepista sordid strains contain or do not contain 500 bp to 800 bp DNA fragments, and determining that the mating types of the protoplast monokaryon of the two to-be-identified Lepista sordid strains are different if the PCR product of the protoplast monokaryon of one of the two Lepista sordid strains contains 500 bp to 800 bp DNA fragments while the PCR product of the protoplast monokaryon of the other of the two Lepista sordid strains does not contain 500 bp to 800 bp DNA fragments.

Description

technical field [0001] The invention relates to a method for identifying or assisting in identifying the monokaryon mating type of the protoplast of the variegated mushroom and the pair of special primers SR-5×16. 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 whethe...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
Inventor 许峰刘宇李登进赵爽王守现王兰青耿小丽
Owner BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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