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Pleurotus tuber-regium protoplast preparation method

A technology of protoplasts and tiger milk mushrooms, applied in the field of bioengineering, can solve problems such as the preparation and application of protoplasts that have not been seen before, and achieve the effect of simple procedures and strong operability

Active Publication Date: 2021-06-22
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are many studies on protoplasts in edible fungi such as ganoderma lucidum, straw mushroom, and oyster mushroom, but there is no research on the preparation and application of protoplasts in tiger milk mushroom

Method used

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  • Pleurotus tuber-regium protoplast preparation method
  • Pleurotus tuber-regium protoplast preparation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Weigh cellulase powder with an enzyme activity of 15000U / g, dissolve it with 0.6mol / L sucrose solution (osmotic pressure stabilizer) to obtain a cellulase solution, make the enzyme activity in the cellulase solution 15U / mL, and Filter through a 0.22 μm sterile microporous membrane, and store at -20°C until use.

[0040] The tiger milk mushroom mycelium fermented to the 4th day was washed 3 times with 0.6mol / L sucrose solution, then β-mercaptoethanol (0.3%, v / v) was added to cover the thalline, and the washed bacterium was treated at room temperature. The mycelium was submerged for 20 minutes, filtered to collect the mycelium, and washed three times with 0.6 mol / L sucrose solution.

[0041] Add 4 times the volume of cellulase solution to the EP tube containing the mycelia, hydrolyze at 30°C for 2.5 hours, then filter with cloth paper membrane, and collect the filtrate. Centrifuge at 4000g for 10min, carefully remove the supernatant, and wash the precipitate (protoplast)...

Embodiment 2

[0044] Weigh the cellulase powder whose enzyme activity is 15000U / g, and dissolve it with 0.6mol / L sucrose solution (osmotic pressure stabilizer) to obtain the cellulase solution, so that the enzyme activity in the cellulase solution is 10U / mL. Filter through a 0.22 μm sterile microporous membrane, and store at -20°C until use.

[0045] The tiger milk mushroom mycelium fermented to the 4th day was washed 3 times with 0.6mol / L sucrose solution, then β-mercaptoethanol (0.3%, v / v) was added to cover the thalline, and the washed bacterium was treated at room temperature. The mycelium was submerged for 20 minutes, filtered to collect the mycelium, and washed three times with 0.6 mol / L sucrose solution.

[0046] Add 5 times the volume of cellulase solution to the EP tube containing the mycelia, hydrolyze at 30°C for 4 hours, then filter with cloth paper membrane, and collect the filtrate. Centrifuge at 4000 g for 10 min, carefully remove the supernatant, and wash the precipitate (p...

Embodiment 3

[0049]Weigh the cellulase powder with enzyme activity of 15000U / g, dissolve it with 0.6mol / L sucrose solution (osmotic pressure stabilizer) to obtain the cellulase solution, make the enzyme activity in the cellulase solution be 30U / mL, and Filter through a 0.22 μm sterile microporous membrane, and store at -20°C until use.

[0050] The tiger milk mushroom mycelium fermented to the 4th day was washed 3 times with 0.6mol / L sucrose solution, then β-mercaptoethanol (0.3%, v / v) was added to cover the thalline, and the washed bacterium was treated at room temperature. The mycelium was submerged for 20 minutes, filtered to collect the mycelium, and washed three times with 0.6 mol / L sucrose solution.

[0051] Add 3 times the volume of cellulase solution to the EP tube containing the mycelium, hydrolyze at 30°C for 2 hours, then filter with cloth paper membrane, and collect the filtrate. Centrifuge at 4000 g for 10 min, carefully remove the supernatant, and wash the precipitate (proto...

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Abstract

The invention relates to a Pleurotus tuber-regium protoplast preparation method, and belongs to the technical field of bioengineering. According to the invention, cellulase is selected for enzymolysis according to the composition of glycosidic bonds of Pleurotus tuber-regium cell wall polysaccharides, Pleurotus tuber-regium protoplasts are effectively obtained, the number can reach 5 * 10 < 6 > / mL, and the regeneration rate reaches 11%. The method provided by the invention has the advantages of simple process and strong operability, and has potential application prospects in the fields of edible and medicinal fungus resource development, genetic breeding, molecular biology, microbiology and the like.

Description

technical field [0001] The invention relates to a method for preparing protoplasts of tiger milk mushroom, belonging to the technical field of bioengineering. Background technique [0002] Tiger Milk Mushroom (Pleurotus tuber-regium), scientific name Pleurotus sclerotium, is a rare edible and medicinal fungus mainly distributed in tropical and subtropical regions. It not only has extremely high nutritional value, but also is rich in various biologically active ingredients. The research on tiger milk mushroom is still mainly in the stage of separation, purification and development of active ingredients, and the research on the synthesis mechanism of active ingredients and the formation mechanism of sclerotia is still rarely involved. Relevant research requires in-depth exploration with the help of molecular biology methods, and the acquisition of protoplasts has become a key step. At present, there are many studies on protoplasts in edible fungi such as ganoderma lucidum, st...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/14C12N1/00C12R1/645
CPCC12N1/14C12N1/005
Inventor 陈磊胡艳红毕黄词张卓敏陈献忠杨海泉
Owner JIANGNAN UNIV
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