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Screening method for high-yielding strain of polyoxin I

A technology of polyoxin and high-yield strains, applied in the field of bioengineering, can solve the problem of low content of polyoxin I components, and achieve the effects of low cost, wide source, and important industrial and agricultural application potential

Pending Publication Date: 2017-10-24
RUSHAN HANWEI BIO TECHN & SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The present invention aims to solve the technical problem that the content of the polyoxin I component in the existing bacterial strains is relatively low, and provides a screening method for high-yield strains of the polyoxin I component, and obtains the polyoxin I group through mutation breeding and screening high-yielding strain

Method used

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  • Screening method for high-yielding strain of polyoxin I
  • Screening method for high-yielding strain of polyoxin I
  • Screening method for high-yielding strain of polyoxin I

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1: Preparation of original strain P0 spores.

[0038] 1. The wild-type P0 of Streptomyces ansochromogenes (the strain purchased from the Chengdu Institute of Biology, Chinese Academy of Sciences) was inoculated on the slant medium from the polyoxin starting strain. Cultivate for 7 days at a temperature of 29° C., select the spores on the slant medium, and add physiological saline to prepare a spore suspension. Incline medium (100ml), according to mass volume ratio, its composition is: Soluble starch: 2g; KNO 3 : 0.1g; K 2 HPO 4 : 0.05g; MgSO 4 ·7H 2 O: 0.05g; NaCl: 0.05g; FeSO 4 ·7H 2O: 0.001g; agar: 2g; adjust the pH value of the medium to 7.0.

[0039] 2. Use the wild-type strain P0 as the starting strain. First, determine the minimum inhibitory concentration of 5-fluorouracil on the P0 strain. Prepare spore culture medium containing different concentrations of 5-fluorouracil (5-fluorouracil concentrations are 0.01, 0.02, 0.05, 0.1, 0.2, 0.3, 0.5, 1 m...

Embodiment 2

[0040] Example 2: Ultraviolet mutagenesis of polyoxin starting strain P0.

[0041] Dilute the spore suspension to 10 7 / mL, take 3mL of the mixed monospore suspension and place it on a sterile plate, and place it in a preheated 20min ultraviolet mutagenesis box for irradiation for 5s-10s. Wherein, the wavelength of the ultraviolet light is 253.5nm, the power of the ultraviolet lamp is 15W, and the vertical distance between the ultraviolet mutagenesis box and the sterile plate is 30cm.

[0042] After the ultraviolet mutagenesis treatment, in order to prevent recovery of the mutation under visible light, the plate was wrapped with black paper and kept in the dark for 2 hours.

[0043] After taking out, carry out serial dilution under red light (10 -1 ~10 -5 ), and then spread them in the order from low concentration to high concentration on spore slant medium plates containing 5-fluorouracil at 3 different concentrations of 0.1mg / mL, 0.3mg / mL and 1mg / mL, and cultivate in the ...

Embodiment 3

[0045] Example 3: Mutagen fermentation screening and fermentation broth analysis.

[0046] Pick 100 mutated single colonies and streak them on a new slant medium plate, and culture them at 28°C for 7 days until the spores grow well. Inoculate 0.5cm 2 Into the seed medium, 250rpm, 29°C, cultivate for 1 day; then inoculate 10% of the inoculum into the fermentation medium for shake flask fermentation, and at the same time use the starting strain as a control, at 29°C, 250rpm shaker, cultivate for 5 days , Stop fermentation at the dying stage.

[0047] Seed culture medium (100ml), according to mass volume ratio, its composition is: yeast extract 1.0g; Tryptone: 1.6g; KNO 3 : 0.1g; K 2 HPO 4 : 0.05g; MgSO 4 ·7H 2 O: 0.05g; NaCl: 0.05g; FeSO 4 ·7H 2 O: 0.001 g; the pH value of the medium is 7.0.

[0048] Fermentation medium (100ml), according to mass volume ratio, its composition is: starch: 5g; soybean meal powder: 2.5g; yeast extract: 0.5g; mannitol: 0.5g; NaCl: 0.5g; 4 ...

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Abstract

The invention relates to a screening method for a high-yielding strain of polyoxin I and solves the technical problem of lower content of polyoxin I in an existing strain. An original strain is Streptomyces ansochromogenes; the method comprises steps as follows: a spore suspension of the original strain is prepared, a 5-fluorouracil-containing resistant plate is coated with the diluted spore suspension subjected to ultraviolet mutagenesis, and after culture, resistant strains are screened and the high-yielding strain of polyoxin I is obtained. Fermentation broth of the screened high-yielding strain of polyoxin I has an inhibition effect on alternaria alternata. The high-yielding strain can be rapidly screened out with the screening method.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to a screening method for polyoxin I component high-yield strains. Background technique [0002] With the continuous advancement of modern biological technology, related agricultural production has a broader development prospect, and at the same time, the demand for high-yield and high-efficiency antibiotics is also increasing. [0003] Polyoxin (polyoxin) is an important nucleoside peptide antibiotic, because its structure is similar to the natural substrate UDP-N-acetylglucosamine of chitin synthase, so it can competitively inhibit chitin Synthesis of substances, thereby inhibiting the growth of fungi. Polyoxin is a safe pesticide with high efficiency, low toxicity and no environmental pollution. It is widely used in agricultural production. It has a good control effect on fungal plant diseases such as sclerotinia and sclerotinia. [0004] In the process of microbial str...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N13/00C12N1/20C12N1/02C12R1/465
CPCC12N1/02C12N1/20C12N13/00
Inventor 李盛英杜磊马圆圆徐晓庆王玉亭
Owner RUSHAN HANWEI BIO TECHN & SCI
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