Preparation method of tilletia controversa protoplasts

A kind of technology of dwarf black powder and protoplast, which is applied in the field of preparation of wheat dwarf black powder protoplast, and can solve the problems of research, pathogenic molecular mechanism of unfavorable pathogenic bacteria and the like.

Pending Publication Date: 2017-08-18
INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

There are no reports on the protoplast preparation system of T. tritici at home and abroad, ...

Method used

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  • Preparation method of tilletia controversa protoplasts
  • Preparation method of tilletia controversa protoplasts
  • Preparation method of tilletia controversa protoplasts

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Embodiment 1: Optimization of the method for preparing T. dwarf Tilletia protoplasts

[0058] Cultivate the teliospores of Tilletia tritici in the soil extract medium at a temperature of 5°C and a relative humidity of 50%. Select the cells of different cultivation days, rinse them with sterilized distilled water, and transfer them to In the T19 liquid culture medium, culture in a full-temperature shaker at 5° C. and 150 rpm to collect the mycelia culture solution, and after filtration, centrifuge at 5000 r / min for 10 minutes to collect the mycelia. Take 1g of mycelium, add 20ml of different combinations of enzyme solutions, 28°C, 180r / min oscillating enzymolysis, every 30min, observe the protoplast acquisition with a microscope, set different experimental parameters, and screen out the most suitable bacteria for protoplast preparation Age, enzymatic hydrolysis time, enzyme system, osmotic pressure stabilizer.

[0059] 1. Bacteria culture time

[0060] Cultivate the te...

Embodiment 2

[0076] Embodiment 2: adopt the optimized method to prepare the protoplast of Tilletia tritici

[0077] A mixed enzyme solution of 1.5% collapse enzyme, 1.5% lysozyme and 1.5% helicase was prepared with 1.2mol / L potassium chloride. Then prepare protoplasts according to the following steps:

[0078] (1) Mycelia culture: Cultivate the teliospores of Triticillium dwarfiae teliospores for 35 days at 5°C and a relative humidity of 50% on the solid medium of the soil extract, and then sterilize the teliospores with sterilized distilled water After rinsing, transfer to T19 liquid culture medium, culture in a full-temperature shaker at 5°C and 150rpm for 10 days, and collect mycelium culture solution.

[0079] (2) Enzymatic hydrolysis of mycelial cell wall: filter the collected mycelial culture solution to remove the liquid medium, centrifuge at 5000r / min for 10min to collect mycelium, take 1g of mycelium, add 20mL of mixed enzyme solution, 28°C, 180r / min, shake After 2.5 hours of enzy...

Embodiment 3

[0082] Embodiment 3: the regeneration of Tilletia tritici protoplast

[0083] After the protoplasts prepared in Examples 1 and 2 were resuspended with 1.2 mol / L of potassium chloride, they were respectively coated in PDA and TB3 solid medium, cultured at 5°C for 2-3 weeks, and the grown cells were observed. colony.

[0084] The result is as Image 6 As shown, the protoplasts of T. tritici grow more single colonies on the TB3 medium, but no single colonies grow on the PDA medium. Therefore, the TB3 medium is more suitable for protoplast regeneration than the PDA medium. It can be used for the regeneration culture of the protoplast of Tilletia tritici.

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Abstract

The invention relates to a preparation method of tilletia controversa protoplasts. The preparation method comprises the following steps: (1) mycelium culture: culturing tilletia controversa teliospore on a soil aqueous extract culture medium, after the teliospore sprouts, transferring the teliospore to a T19 liquid culture medium and carrying out shaking culture, and collecting mycelium, wherein the sum of the number of culture days on the soil aqueous extract culture medium and the number of shaking culture days on the T19 liquid culture medium is 40-50 days; and (2) cell wall cracking: taking a KCl solution as an osmotic pressure stabilizer, preparing mixed enzyme liquid, adding the mixed enzyme liquid in the mycelium, and carrying out shaking enzymolysis for 2-3 hours at the temperature of 28 DEG C to obtain the tilletia controversa protoplasts. The mixed enzyme liquid comprises the following components in percentages by mass: 1.5% of driselase, 1.5% of lywallzyme and 1.5% of helicase. The yield of the tilletia controversa protoplasts prepared by the method reaches up to 12.0* 105 per mL, and the released protoplasts are uniformly distributed in a scattered manner and cannot be accumulated or piled.

Description

technical field [0001] The invention relates to the technical field of preparation and regeneration of fungal protoplasts in cell engineering, in particular to a method for preparing protoplasts of T. dwarfiae. Background technique [0002] Wheat dwarf bunt disease (DB) is a quarantine disease caused by Tilletia controversa Kühn (TCK), which has the characteristics of serious damage and difficulty in control. With devastating damage. Susceptible plants usually have the symptoms of dwarfing and multiple tillers, and the grains on the ear turn into black powder, which is the teliospore of the pathogen. The teliospore has a fishy smell, which causes the yield and quality of wheat to decrease. At home and abroad, the research on wheat dwarf smut mainly focuses on the identification, biological characteristics and molecular detection technology of the pathogen, and there are few studies on the pathogenic mechanism of the pathogen. Genetic transformation technology is an importa...

Claims

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Application Information

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IPC IPC(8): C12N1/14C12R1/645
CPCC12N1/14
Inventor 高利沈慧敏陈万权刘太国刘博
Owner INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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