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Gibberella fujikuroia mutant strain with high yield of gibberellin GA3 and applications thereof

A technology of gibberellin and gibberellin, which is applied in the field of mutant strains of gibberellin fujikura, can solve the problems of unreported high-yield gibberellin gibberellin fujikura and its application, and is beneficial to solid-liquid separation and fermentation The effect of level improvement and production cost reduction

Active Publication Date: 2019-07-23
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

China's invention patent (publication number CN104892554A) has described about gibberellin GA 3 However, there is no report of Gibberella Fujikura with high yield of gibberellin and its application. The yield of traditional strains has been hovering between 1.8g and 2g, which needs to be further improved

Method used

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  • Gibberella fujikuroia mutant strain with high yield of gibberellin GA3 and applications thereof
  • Gibberella fujikuroia mutant strain with high yield of gibberellin GA3 and applications thereof
  • Gibberella fujikuroia mutant strain with high yield of gibberellin GA3 and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Embodiment 1: preparation of wild Gibberella fujikura protoplast

[0036] (1) Preparation of the slant: inoculate the wild Gibberella fujikura strain into the slant medium, culture at 28° C. for 3 to 7 days, and transfer to the seed solution after the aerial hyphae cover the slant.

[0037] (2) Preparation of seed solution:

[0038] Pick a small piece of bacteria in the step (1) and inoculate it into the seed medium, and cultivate it at 28° C. and 250 rpm for 48 hours to obtain the seed liquid. The seed medium was prepared as follows: corn starch 20g / L, sucrose 20g / L, peanut powder 20g / L, soybean powder 20g / L, potassium dihydrogen phosphate 1.0g / L, magnesium sulfate 1.0g / L, the solvent was Tap water with natural pH, sterilized at 121°C for 30 minutes.

[0039] (3) Fermentation culture

[0040] 40mL of fermentation medium was loaded into a 250mL shake flask, and the seed solution was inoculated at a volume concentration of 1-4% during fermentation, and fermented at 28...

Embodiment 2

[0043] Example 2: High Yield GA 3 Screening of Mutants

[0044] (1) Inoculate the wild-type Gibberella fujikura into the slant medium, cultivate at 28°C for 3 to 7 days, inoculate a small bacterial block from the slant into the YEPD liquid medium (yeast extract 0.3%, peptone 1%, glucose 2%) , the solvent is water), wash once with sterile water and salt buffer, take a small amount of mycelium and add wall-breaking buffer, put it in a water-bath shaker at 30°C for 45 minutes, centrifuge at 3000rpm for 10min, discard the supernatant and wash with sterile water Add buffer to suspend once.

[0045] (2)Co 60 Mutagenesis method: spread the activated bacterial strain in step (1) on MYG solid medium (0.5% maltose, 0.5% yeast powder, 1% glucose, 5M sucrose, 2% agar), and cultivate at 28°C for 3-7 days up to Go out bacterial colony, prepare protoplast by step (1), control bacterium number about 10 7 Individuals / mL, Co 60 Mutagenesis was performed on the protoplasts. Will pass throu...

Embodiment 3

[0055] Example 3: 18S rDNA molecular identification

[0056] (1) DNA extraction: Centrifuge the mycelia, put a small amount into a 1.5mL EP tube, add 978μL sodium phosphate buffer to suspend the bacteria; transfer the bacterial suspension to a Lysing Matrix E Tube, add 112μL MTBuffer and mix well; use MP Break the cells with a homogenizer, set the speed to 6.0, and set the time to 40s; centrifuge the Lysing Matrix E Tube at 12,000 rpm for 10 minutes to remove sample debris; transfer the supernatant to a new EP tube, add 250 μL of PPS reagent, and hold Shake the centrifuge tube 10 times to mix well, centrifuge at 12000rpm for 5min; transfer the supernatant to a clean 10mL centrifuge tube, add 1mL Binding Matrix suspension, turn the centrifuge tube upside down for 2min, let it stand for 3min, make the DNA attach to the Binding Matrix, and Wait for the silica matrix to precipitate; carefully remove 500 μL of the supernatant (avoid aspiration of the precipitate), re-mix the remai...

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Abstract

The invention discloses Fusarium fujikuroi GA-251 and applications thereof, wherein the Fusarium fujikuroi GA-251 is a Gibberella fujikuroia mutant strain and has high yield of gibberellin GA3. The Fusarium fujikuroi GA-251 is preserved in China Center for Type Culture Collection, wherein the address is Wuhan University, Wuhan, China, the postal code is 430072, the preservation number is CCTCC NO:M 2019201, the preservation date is March 25th, 2019. The strain is obtained by carrying out radiation composite mutagenesis on protoplasts through lithium chloride and cobalt 60, and gibberellin GA3production of the strain is improved by 20%. The method has the advantages of high titer and easy extraction of the final product, and overcomes the defects of low fermentation titer and high cost oforiginal processes.

Description

[0001] (1) Technical field [0002] The invention relates to a high-yield gibberellin GA 3 Mutant strain of Gibberella fujikura—Bakanae oryzae GA-251 and its application. [0003] (2) Background technology [0004] Gibberellins (Gibbeerllins, GAs) is a plant hormone, widely used in agriculture, forestry, horticulture, food brewing and many other fields. Its preparation methods mainly include plant extraction method, chemical method, and microbial fermentation method. However, microbial fermentation method has broad industrial application prospects because of its low production cost and low environmental pollution. Mycin was first discovered by Japanese plant pathologist Kenkichi Sawada in the study of rice bakanae disease, which was later confirmed by his colleague Eiichi Kurosawa, who published a paper in 1926 showing that the symptoms of this disease can be obtained by Applying sterile fungal cultures to replicate, he found that the secretions also stimulated the growth of ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/14C12P27/00C12R1/77
CPCC12P27/00C12N1/145C12R2001/77
Inventor 柳志强张博蒋欢郑裕国
Owner ZHEJIANG UNIV OF TECH
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