Method and special primer SR-5*13 for differentiating and auxiliarily differentiating mating types of bioplast monokaryons of lepista sordida (Fr) sings

A technology of protoplasts and variegated mushrooms, applied in biochemical equipment and methods, botany equipment and methods, microbial measurement/inspection, etc., can solve the problems of narrow temperature range, poor insect resistance, low yield, etc., to achieve Accurate and reliable results

Active Publication Date: 2013-07-03
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,

Method used

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  • Method and special primer SR-5*13 for differentiating and auxiliarily differentiating mating types of bioplast monokaryons of lepista sordida (Fr) sings
  • Method and special primer SR-5*13 for differentiating and auxiliarily differentiating mating types of bioplast monokaryons of lepista sordida (Fr) sings
  • Method and special primer SR-5*13 for differentiating and auxiliarily differentiating mating types of bioplast monokaryons of lepista sordida (Fr) sings

Examples

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-5×13 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-5×13, 10×Taq buffer, dNTP mix, Taq DNA polymerase and ddH 2 O composition.

[0049] Among them, the PCR primer pair SR-5×13 is composed of two single-stranded DNAs, SR-5×13-pre-F and SR-5×13-pre-R, and its sequence is as follows:

[0050] SR-5×13-pre-F:5'-ATCAGGTAGGTAGGCGTAA-3' (SEQ ID No.1)

[0051] SR-5×13-pre-R: 5'-CCGTTAAAGCCAGTCTAAAC-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] Inoculate...

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Abstract

The invention discloses a method and a special primer SR-5*13 for differentiating and auxiliarily differentiating mating types of bioplast monokaryons of lepista sordida (Fr) sings. The method comprises the following steps: genome DNAs of bioplast monokaryons of two lepista sordida (Fr) sings to be differentiated are respectively taken as templates; PCR primers shown by SEQ ID No. 1 and SEQ ID No. 2 are used for PCR increase; the sizes of the obtained PCR products are detected; if the PCR products of the bioplast monokaryons of both the two lepista sordida (Fr) sings to be differentiated contain DNA fragments of 500bp-800bp or don't contain the DNA fragments of 500bp-800bp, the mating types of the bioplast monokaryons of the two lepista sordida (Fr) sings are same; and if the PCR product of the bioplast monokaryon of one of the two lepista sordida (Fr) sings contains DNA fragments of 500bp-800bp, and the PCR product of the bioplast monokaryon of the other of the two lepista sordida (Fr) sings dose not contain the DNA fragments of 500bp-800bp, the mating types of the bioplast monkaryones of the two lepista sordida (Fr) sings are different.

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-5×13. 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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IPC IPC(8): C12Q1/68C12N15/11A01H1/04
Inventor 许峰刘宇赵爽李登进王守现耿小丽王兰青
Owner BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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