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Method for identifying or assisting in identifying mating types of Lepista sordid protoplast monokaryons and special primer pairs IS-873 thereof

A technology of protoplasts and variegated mushrooms, applied in biochemical equipment and methods, measurement/testing of microorganisms, DNA/RNA fragments, etc., can solve the problems of narrow temperature range, poor insect resistance, low yield, etc., and achieve accurate high performance and reliability

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, narrow temperature range, fragile fruiting bodies, and difficult transportation and storage.

Method used

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  • Method for identifying or assisting in identifying mating types of Lepista sordid protoplast monokaryons and special primer pairs IS-873 thereof
  • Method for identifying or assisting in identifying mating types of Lepista sordid protoplast monokaryons and special primer pairs IS-873 thereof
  • Method for identifying or assisting in identifying mating types of Lepista sordid protoplast monokaryons and special primer pairs IS-873 thereof

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 IS-873 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 IS-873, 10X Taq buffer, dNTP mix, Taq DNA polymerase and ddH 2 O composition.

[0049] Among them, the PCR primer pair IS-873 is composed of two single-stranded DNAs, IS-873-pre-F and IS-873-pre-R, and its sequence is as follows:

[0050] IS-873-pre-F:5'-GACAGACAGACAGACAAGT-3' (SEQ ID No.1)

[0051] IS-873-pre-R: 5'-GACAGACAGACAGACAGAC-3' (SEQ ID No. 2).

[0052] 10X 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 the 3...

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Abstract

The invention discloses a method for identifying or assisting in identifying the mating types of Lepista sordid protoplast monokaryons and special primer pairs IS-873 thereof. The method for identifying or assisting in identifying the mating types of the Lepista sordid protoplast monokaryons comprises the following steps: respectively taking the genome DNA of two Lepista sordid protoplast monokaryons to be identified as templates; performing polymerase chain reaction (PCR) amplification on by using PCR primer pairs shown in SEQ ID No. 1 and 2; detecting the size of the obtained PCR products; if the PCR products of the two Lepista sordid protoplast monokaryons to be identified comprise 500 to 600 bp of DNA segments or do not comprise 500 to 600 bp of DNA segments, the mating types of two Lepista sordid protoplast monokaryons are identical; and if the PCR product of one of the two Lepista sordid protoplast monokaryons to be identified comprises 500 to 600 bp of DNA segments and the PCR product of the other one of the two Lepista sordid protoplast monokaryons to be identified does not comprise 500 to 600 bp of DNA segments, the mating types of two Lepista sordid protoplast monokaryons are different.

Description

technical field [0001] The invention relates to a method for identifying or assisting in identifying the monokaryon mating type of protoplasts of variegated mushrooms and the special primer pair IS-873. 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 indiv...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
Inventor 许峰刘宇赵爽李登进王守现王兰青耿小丽
Owner BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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