Method for breeding gibberellic acid high-producing strains by performing low-energy ion induced mutation on gibberella

A technology of high-yield strains and low-energy ions, applied in the field of microbial mutagenesis, to achieve the effects of large-scale industrial production, good mutagenesis effect, and stable high-yield traits

Inactive Publication Date: 2015-03-04
ZHENGZHOU UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the low-energy ion beam implantation method is used for the selection and breeding of gibberellic acid production strains, and there are no relevant patents and literature reports.

Method used

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  • Method for breeding gibberellic acid high-producing strains by performing low-energy ion induced mutation on gibberella

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Experimental program
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Effect test

Embodiment 1

[0025] Get Gibberella strain and make 10 with sterile water 8 protoplast suspension per mL; take 0.1mL of protoplast suspension and evenly spread it on a sterile flat petri dish, and dry it in a sterile state; use energy 10keV, dose 1.5×10 14 ions / cm 2 Nitrogen ion beam, pulse injected into the air-dried protoplast, add 1mol / L sorbitol solution 10 times ice bath, the sorbitol solution used contains 10mmol / L Tris-Cl, pH7.5; 50mmol / L CaCl 2 . After suspending, spread it on the PDAS regeneration medium plate and culture it at 28°C for 5 days. The PDAS regeneration medium used contains 20% potato juice, 2% glucose, 3.5%~4.1% NaCl, 1.5%~2% agar, pH6. 0~6.5; select the cultured single strain with white round shape, normal shape, vigorous growth and diameter of 2~4mm to inoculate in a 500mL shaker flask filled with 100mL liquid fermentation medium, and culture it in a shaker at 28°C and 220rpm for 180h. 40 mutagenic single strains were randomly selected as the object of investiga...

Embodiment 2

[0028] Get Gibberella strain and make 10 with sterile water 9 protoplast suspension per mL; take 0.3mL protoplast suspension and evenly spread it on a sterile flat petri dish, and dry it under aseptic condition; use energy 20keV, dose 1.5×10 15 ions / cm 2 Nitrogen ion beam, pulse injected into the air-dried protoplast, add 1mol / L sorbitol solution 10 times ice bath, the sorbitol solution used contains 10mmol / L Tris-Cl, pH7.5; 50mmol / L CaCl 2 . After suspending, spread it on the PDAS regeneration medium plate and culture it at 28°C for 5 days. The PDAS regeneration medium used contains 20% potato juice, 2% glucose, 3.5%~4.1% NaCl, 1.5%~2% agar, pH6. 0~6.5; Select a single strain with a white round shape, normal shape, vigorous growth, and a diameter of 2~4mm to inoculate in a 500mL shaker flask containing 100mL of liquid fermentation medium, and culture it in a shaker at 28°C and 220rpm for 180h. 40 mutagenic single strains were randomly selected as the object of investigati...

Embodiment 3

[0031] The gibberellic acid-yielding gibberellic acid-producing gibberellic acid mutant strain obtained by mutagenesis was continuously passaged on the PDA slant medium (ingredients: 200g potato, 20g glucose, 15g agar, 1000mL tap water) for 12 consecutive passages. times, after each passage in the fermentation medium (ingredients: liquefied starch 100g / L, peanut cake powder 15g / L, soybean meal powder 3g / L, magnesium sulfate 0.75g / L, potassium dihydrogen phosphate 1.0g / L, pH 5~6) Medium culture, determination of gibberellic acid content, shake flask fermentation results see figure 1 . The content of gibberellic acid produced by fermentation of the original strain was 100%, compared with other generations, the result was that the amount of gibberellic acid produced after 10 passages was still stable, maintaining above 93%, indicating that the genetic stability of the mutant strain was better, Can be used as a potential production strain.

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Abstract

The invention discloses a method for breeding gibberellic acid high-producing strains by performing low-energy ion induced mutation on gibberella. The method comprises the following steps: firstly, preparing a protoplast suspension of 108-109 / mL by use of a gibberella strain and sterile water; applying the protoplast suspension to a culture dish for air drying; injecting a pulsed nitrogen ion beam into the air-dried protoplasts to induce the mutation of the protoplasts; adding the mutated protoplasts to sorbitol solution ice bath for suspending, and then applying the suspension to a PDAS regeneration medium plate for cultivation; performing inoculation of the well growing individual strains into a shake flask filled with a liquid fermentation culture medium for shake cultivation, thereby obtaining the gibberellic acid high-producing strains. The method for breeding the gibberellic acid high-producing strains by performing low-energy ion induced mutation on the gibberella has the advantages that the mutation of the gibberella is induced in a pulsed nitrogen ion beam injection manner and the high-producing mutant gibberella strains can be bred; according to the method, the direct mutation rate of the mutant strains can be 10%-40%, and the fermentation titer is increased by 30-90% in contrast with that of the original strain.

Description

technical field [0001] The invention relates to microbial mutagenesis technology, in particular to a method for breeding gibberellic acid high-yield strains by using low-energy ions to mutagen gibberella. Background technique [0002] Gibberellic acid (gibberellic acid) is a plant growth regulator belonging to diterpenoids. It has the functions of promoting seed germination and plant growth, early flowering and fruiting, etc., and has obvious promotion effects on the yield and quality of crops and vegetables. It is widely used in agricultural production and brewing industry. With the promotion of agricultural technology and the expansion of vegetable and fruit planting, the demand for gibberellic acid has increased rapidly, and the production volume cannot meet the actual demand of the market. [0003] Gibberellic acid is mainly produced by fermentation of Gibberella fujikuroi. The production of gibberellic acid has a history of half a century, and the improvement of strai...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/01C12R1/645
Inventor 张一折张书杰朱朝印马轩刘建华方业忠
Owner ZHENGZHOU UNIV
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