Phellinus linteus strain and breeding method thereof

A technology of Phellinus linteus and strains, which is applied in the fields of biotechnology and microbial mutation breeding, can solve problems such as no Phellinus linteus strains and the like, and achieve the effect of increasing the yield of biomass and metabolic active substances

Active Publication Date: 2016-09-28
SHANGHAI ACAD OF AGRI SCI
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  • Phellinus linteus strain and breeding method thereof
  • Phellinus linteus strain and breeding method thereof
  • Phellinus linteus strain and breeding method thereof

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Embodiment 1

[0030] Embodiment 1: Preparation and mutagenesis of protoplasts

[0031] (1) Activation and cultivation of strains Transfer the slant strain SH1 to a PDA plate, and culture in a 26°C incubator for 8-10 days. Pick the plate mycelium into a homogenizer, add a small amount of liquid culture medium, homogenize and crush it, inoculate it in 100mL liquid culture medium, keep the temperature at 26°C, and culture it statically for 10 days, during which the mycelia are slightly shaken 2-3 times a day.

[0032] (2) Preparation of protoplasts The hyphae were collected by filtration and cultured statically, rinsed with sterile water for 1-2 times, and the water on the surface of the mycelia was blotted dry with sterile absorbent paper. Add 1mL enzyme solution (0.12g wall-lyzing enzyme (purchased from Guangdong Institute of Microbiology), 0.03g disintegrating enzyme (purchased from Sigma) to 12mL 0.6mol / L mannitol solution according to every 0.3g mycelium, and after 0.2 μm bacteria filter...

Embodiment 2

[0034] Embodiment 2: Screening of mutagenic strains

[0035] (1) Preliminary screening of mutagenized strains Take 100 μL of the bacterial solution after mutagenesis and evenly spread it on the regeneration medium. After culturing in a 26°C incubator for 7-10 days, the mutagenized strains will be regenerated. Densely mutated strains were quantitatively inoculated on a new PDA plate with a hole puncher with a diameter of 0.6 cm. After culturing for 10 days, the mycelium growth rate was counted. Taking the average growth rate of 30 non-mutated protoplast regenerated strains as a control, select the mutagenized strain whose mycelial growth rate is greater than that of the starting strain, and carry out the subculture experiment. The growth rate of mycelium was measured in each generation, passed down for 5 times, and 10 excellent strains with fast mycelial growth rate and stable genetics were selected.

[0036] (2) Re-screening of mutagenized strains Take the 10 excellent strain...

Embodiment 3

[0045] Embodiment 3: Identification and yield determination of mutagenized strains

[0046] (1) Strain mutagenesis identification

[0047] Antagonism experiment: Antagonism experiment is a method that can quickly identify genetic differences between strains. It can not only be used to determine the relationship between strains of different species, but also to identify whether strains of the same species have mutated. The results showed that the mutagenized strain A67 produced obvious antagonism with the original starting strain SH1, and it was confirmed that the genetic material of the strain had changed from the aspects of hyphal morphology.

[0048] RAPD random primer amplification:

[0049] Using the CTAB method to extract the DNA of the mutagenized strain and the starting strain, 12 primers with good repeatability were screened out from 52 RAPD primers, which could reflect the differences between bacterial strains (see the primer sequence for details). figure 2 ).

[...

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Abstract

The invention provides a phellinus linteus strain and a breeding method of the phellinus linteus strain. The breeding method comprises the steps of preparation and mutagenesis of protoplast, screening of a mutagenesis strain, and verification of the mutagenesis strain. The shake flask biomass of the phellinus linteus strain obtained through the breeding method reaches 17.92 g/L and is higher than that of an original strain by 9.51%. The yield of flavone reaches 0.47 g/L and is higher than that of the original strain by 52.16%, and the yield of polysaccharide reaches 3.75 g/L and is higher than that of the original strain by 67.95%. The biological yield and the yield of metabolic active matter are also remarkably higher than those of the parent strain SH1, and the free radical scavenging capability is remarkably improved.

Description

technical field [0001] The invention belongs to the fields of biotechnology and microbial mutation breeding, and in particular relates to a Phellinus phylloxera strain and a breeding method thereof. Background technique [0002] Phellinus is a large and rare medicinal fungus, which belongs to Fungi Kingdom, Basidiomycota, Agaricaceae, and Prussaceae. Phellinus has physiological functions such as tumor inhibition, anti-aging, anti-oxidation, and enhancing cellular immunity. Its main active substances are polysaccharides and small-molecule flavonoids. With the improvement of social and economic development level, the demand for Phellinus Phellinus in domestic and foreign markets has increased sharply, and the resources of wild Phellinus Phellinus are extremely limited. Large-scale production of Phellinus Phellinus mycelia by liquid fermentation technology is an effective way to meet the needs of domestic and foreign markets. , and the yield and activity of Phellinus mycelia o...

Claims

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Application Information

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IPC IPC(8): C12N1/14C12N13/00C12N15/01C12R1/645
CPCC12N13/00C12N15/01C12N1/145C12R2001/645
Inventor 杨焱曲德辉唐传红张赫男张劲松冯杰刘艳芳吴迪颜梦秋周帅冯娜唐庆九张忠
Owner SHANGHAI ACAD OF AGRI SCI
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