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Preparation and regeneration method of pythium oligandrum protoplasts and composite enzymatic hydrolysate

A compound enzymatic solution and protoplast technology, applied in the field of microbial protoplast preparation and regeneration, can solve the problems of inability to complete the preparation of Pythium oligandrum, the inability to remove the cell wall of Pythium oligandrum, and the difference in cell wall components, so as to achieve regeneration rate High, maintain integrity, good stability

Active Publication Date: 2016-11-16
周剑平 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The cell wall composition of different strains is significantly different, so the compound enzymatic solution used to remove the cell wall in the preparation of protoplasts is also very different. At present, there is no special compound enzymatic solution for Pythium oligandrum, and the compound enzymatic solution used by other strains cannot The cell wall of Pythium oligandrum is removed so that the preparation of Pythium oligandrum protoplasts cannot be completed

Method used

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  • Preparation and regeneration method of pythium oligandrum protoplasts and composite enzymatic hydrolysate
  • Preparation and regeneration method of pythium oligandrum protoplasts and composite enzymatic hydrolysate
  • Preparation and regeneration method of pythium oligandrum protoplasts and composite enzymatic hydrolysate

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1 Preparation and regeneration of Pythium oligandrum protoplasts.

[0030] Inoculate Pythium oligandrum hyphae discs on PDA plate medium, culture at 26°C for 4 days, and scrape off all the mycelium and spore mixture on the 4 plates with a sterile inoculation loop (about 1g ), placed in a centrifuge tube; with 5mL osmotic pressure stabilization solution (0.8M mannitol, 25mM CaCl 2 , 10mM Tris-HCl pH7.5) wash the mixture of mycelia and spores, 5500rpm, centrifuge for 10min, repeat the washing and centrifugation for 3 times, and collect the mixture of mycelia and spores of Pythium oligandrum; the mixture of mycelia and spores of Pythium oligandrum collected in the previous step Add compound enzymatic hydrolysis solution (4mL 1%lysing enzyme + 4mL1%cellulase+200U lyticase), fully dissolve for enzymolysis, 30℃, 80rmp, enzymolysis for 2h, to obtain Pythium oligandrum protoplasts; The mixture of mycelium protoplasts and mycelial spore residues was filtered through 4 l...

Embodiment 2

[0033] Example 2 The composition determination experiment of the compound enzymatic hydrolyzate.

[0034] According to the method described in Example 1, different ratios of lysing enzyme, cellulase, and lyticase were used to enzymolyze the mixture of Pythium oligandrum mycelium spores, collect protoplasts, measure the concentration of protoplasts, and perform protoplast regeneration experiments to determine the protoplasts Regeneration rate, according to the experimental results to determine the best composition of the compound enzymatic hydrolyzate, the experimental results are shown in the table below.

[0035]

[0036] It can be seen from experiments that using one of lysing enzyme, cellulase or lyticase alone (such as ⑴~⑶), or enzymatically cleaving Pythium oligandrum mycelia without the participation of lyticase (such as ⑷), cannot make oligoandrum Pythium male releases protoplasts. When the activity of lyticase added to 1g of mycelia exceeds 200U (such as ⑿), it will ...

Embodiment 3

[0037] Example 3 Determination of the enzymatic hydrolysis time of the compound enzymatic hydrolyzate.

[0038] Using the composite enzymolysis solution determined in Example 2, i.e. 4mL 1%lysing enzyme+4mL1%cellulase+200U lyticase, respectively enzymatically hydrolyze the Pythium oligandrum mycelium spore mixture for 1h, 1.5h, 2h, 2.5h, 3h, 4h, 5h and overnight, and the rest of the method is the same as in Example 1, and the preparation of protoplasts is observed.

[0039] The experimental results showed that after 1 hour and 1.5 hours of enzymatic hydrolysis, there were more bacteria that had not been enzymatically hydrolyzed, and less protoplasts were released; Not good; after overnight enzymatic hydrolysis, the protoplasts were all enzymatically hydrolyzed into debris residues; after 2-3 hours of enzymatic hydrolysis, a large number and high activity of Pythium oligandrum protoplasts could be obtained.

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Abstract

The invention provides a preparation and regeneration method of pythium oligandrum protoplasts. The reparation and regeneration method comprises the following steps: a, preparing thallospore hybrids; b, preparing protoplasts; c, purifying the protoplasts; and d, regenerating the protoplasts. According to the method, a complex enzyme system containing lysing enzyme, cellulsase and lyticase is adopted for carrying out enzymolysis on the mycelia of pythium oligandrum, then the pythium oligandrum protoplasts are obtained, and the protoplasts are regenerated through liquid shaking culture adopting a regeneration culture medium taking mannitol as an osmotic pressure stabilizer. The number of the pythium oligandrum protoplasts prepared by adopting the method is large, the yield is high, and the activity is good; through the liquid culture regeneration path, relatively high regeneration rate can be obtained, and the regeneration rate can achievce 1.25%. The method is easy and convenient to operate and good in stability, the obtained pythium oligandrum protoplasts can completely satisfy the research and application such as late gene genetic transformation and cell fusion. The invention further provides composite enzymatic hydrolysate for preparing the pythium oligandrum protoplasts, the composite enzymatic hydrolysate is a key factor in the preparation process of the pythium oligandrum protoplasts, and the enzymatic hydrolysate is suitable for being further developed and applied.

Description

technical field [0001] The invention relates to a method for preparing and regenerating microbial protoplasts, in particular to a method for preparing and regenerating protoplasts of the biocontrol fungus Pythium oligandrum. The invention also relates to a compound enzymolysis solution for preparing Pythium oligandrum protoplast. technical background [0002] Pythium oligandrum belongs to the genus Pythium of the family Pythium Oomycota. It is a soil-dwelling saprophytic fungus. It can colonize the root circle of a variety of important crops. It is non-toxic to plants and animals and is environmentally safe. The hyphae of Pythium oligandrum can parasitize the pathogenic bacteria, interfere with the metabolic activities of the host, consume nutrients in the cells, and cause the death of the pathogens. At the same time, they can also synthesize tryptamine, tryptophan, indole acetic acid and other growth active substances to promote Plant physiological metabolism, nutrient upt...

Claims

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Application Information

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IPC IPC(8): C12N1/14C12N1/06C12R1/645
CPCC12N1/06C12N1/14
Inventor 周剑平王沛雅祝英王治业彭轶楠巩晓芳杨晖
Owner 周剑平
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