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Breeding method of strain with high yield of phenazine-1-formamide based on genome shuffling

A technology of genome shuffling and high-yield strains, applied in the field of bioengineering, can solve the problems of phenazine-1-carboxamide high-yield strain breeding that have not yet been reported, difficult work, chromosome damage, etc., to save labor costs and improve efficiency , the effect of simple operation

Inactive Publication Date: 2017-02-15
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there are no relevant reports on the breeding of phenazine-1-carboxamide high-yielding strains using this technology
In addition, the traditional genome shuffling method needs to mark the two parents before fusion in advance, including integration markers, genetic defect markers, drug resistance markers, etc., and then screen the fusions according to the markers, and finally select high-yielding strains from the fusions. , so the steps of this method are cumbersome, the work is difficult, the implementation period is long, and some markers may cause damage to the chromosome

Method used

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  • Breeding method of strain with high yield of phenazine-1-formamide based on genome shuffling
  • Breeding method of strain with high yield of phenazine-1-formamide based on genome shuffling
  • Breeding method of strain with high yield of phenazine-1-formamide based on genome shuffling

Examples

Experimental program
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Effect test

Embodiment 1

[0034] 1.1 NTG mutagenesis and primary screening

[0035] In this example, Pseudomonas chloropinus HT66 was used as the starting strain, and a high-yield mutant strain was obtained by treatment with a chemical mutagen. The mutagenesis treatment may be NTG mutagenesis, but is not limited to this method. When using NTG mutagenesis treatment, the steps are as follows:

[0036]Prepare a NTG mother solution with a concentration of 10g / L and dilute it proportionally when used. Aspirate 1mL logarithmic phase bacterial suspension, wash the bacterial cells twice with sterile 0.01mol / L PBS buffer (pH7.4); The concentration was 50, 100, 200, 500, 1000g / L, and treated at 28°C for 20min; adding 0.5mL of 1mol / L NaCl solution to terminate the reaction, using sterile medium to wash the bacteria for 3 times to completely remove residual NTG, diluting appropriately, and then applying After 24 hours, count the number of colonies grown on the plate to obtain the lethal rate. When the dose o...

Embodiment 2

[0078] 2.1 Cobalt-60γ-ray mutagenesis and primary screening

[0079] In this example, Pseudomonas chloropinus HT66 was used as the starting strain, and a high-yield mutant strain was obtained by physical mutagenesis. The mutagenesis treatment may be cobalt-60γ-ray mutagenesis, but is not limited to this method. The specific method includes the following steps:

[0080] 1. Take out the hair strain HT66, activate and cultivate it as seed solution.

[0081] 2. Take the seed solution and centrifuge, suck off the culture medium, and resuspend the bacteria with PBS buffer to make the cell concentration about 10 7 to 10 8 individual / mL.

[0082] 3. Divide the cell suspension into sterilized glass sampling vials, and place them in a cobalt-60 gamma ray radiation environment to receive irradiation; the vials of the control bacteria solution do not receive irradiation.

[0083] The absorbed doses received by the cell suspensions in each group were 0.225, 0.320, 0.430, 0.490 and 0...

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Abstract

The invention relates to a breeding method of a strain with high yield of phenazine-1-formamide based on genome shuffling. The breeding method comprises the following steps: by taking pseudomonas chlororaphis HT66 as an initial strain, carrying out mutagenesis treatment, thus obtaining a genome library containing various different positive mutations, and screening out a plurality of mutant strains with high PCN yield; and carrying out protoplast preparation and protoplast fusion on the mutant strains, and screening out fusants with high PCN yield. The breeding method has the beneficial effects that the mutant strains of pseudomonas chlororaphis HT66 are screened by adopting the method combining mutagenesis treatment with the genome shuffling technology, the relevant parameters of protoplast preparation and fusion are optimized, the genome shuffling efficiency is improved, the strain with high yield of phenazine-1-formamide is obtained, and compared with that of the initial strain, the yield of phenazine-1-formamide of the obtained strain is improved by 2.8 times to the maximum. The method provided by the invention is easy to operate, and the parent strain does not need to be labeled, so that the labor cost is greatly reduced, and the screening period is shortened.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to a breeding method for high-yield strains of phenazine-1-carboxamide based on genome shuffling. Background technique [0002] Phenazine-1-carboxamide (PCN) is an effective new biopesticide with broad-spectrum inhibitory effect on phytopathogenic fungi. Pseudomonas chlororaphis HT66 (Pseudomonas chlororaphis HT66)CCTCCNO:M2013467 is a strain of Pseudomonas chlororaphis that produces phenazine-1-carboxamide (PCN) isolated from rice rhizosphere soil. It is very safe. And the yield of PCN is as high as 420mg / L, which is the wild strain with the highest yield of phenazine compounds reported in the world. The bacterium has a strong ability to secrete phenazine compounds, and can quickly transport the phenazine compounds synthesized in the cell to the outside of the cell, forming green crystals in the medium, which not only reduces the feedback inhibition phenomenon in metabolic ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12N15/01C12N15/03C12N1/02C12P17/12C12R1/38
CPCC12N1/20C12N1/02C12N15/01C12N15/03C12P17/12
Inventor 彭华松张雪洪米彦卿
Owner SHANGHAI JIAO TONG UNIV
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