Fusarium fujikuroi mutant strain bred through ARTP mutagenesis technique and application

A technology of Gibberella Fujikura and mutant strains, which is applied in the field of Gibberella Fujikura mutant strains, can solve the problems of irritation, poor genetic stability of strains, flammability, etc., achieve significant economic and social benefits, and is beneficial to solid-liquid Separation, the effect of reducing production costs

Active Publication Date: 2019-12-03
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

After long-term physical and chemical mutagenesis, the production of Gibberella will be retained, and chemical mutagenesis has disadvantages such as environmental pollution and dangerous operation. For example, nitrosoguanidine, as a mutagen for microbial breeding, is flammable, toxic, carcinogenic, and irritating. chara

Method used

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  • Fusarium fujikuroi mutant strain bred through ARTP mutagenesis technique and application
  • Fusarium fujikuroi mutant strain bred through ARTP mutagenesis technique and application
  • Fusarium fujikuroi mutant strain bred through ARTP mutagenesis technique and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Embodiment 1: preparation of wild Gibberella fujikura protoplast

[0040] (1) Preparation of the slant: inoculate the wild Gibberella fujikura strain (preserved in the laboratory) 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.

[0041] (2) Preparation of seed solution:

[0042] 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.

[0043] (3) Fermentation culture

[0044] 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 fer...

Embodiment 2

[0047] Example 2: High Yield GA 3 Screening of Mutants

[0048] (1) Inoculate the wild-type Gibberella fujikura into the slant medium, cultivate it for 3-7 days at 28°C, and inoculate a small piece of bacteria from the slant into the YEPD liquid medium (yeast extract 0.3%, peptone 1%, glucose 2%) ), wash once with sterile water and salt buffer solution, take a small amount of mycelium and add wall-breaking buffer solution, place it in a water-bath shaker at 30°C for 45 minutes, centrifuge at 3000rpm for 10 minutes, discard the supernatant and wash once with sterile water, then add buffer liquid suspension.

[0049] (2) ARTP mutagenesis method: apply the activated bacterial strain of step (1) on MYG solid medium (maltose 0.5%, yeast powder 0.5%, glucose 1%, sucrose 5M, agar 2%), and cultivate for 3- 7 days to grow bacterium colony, prepare protoplast by step (1), the control bacterium number is about 10 7 individual / mL.

[0050] Before using the ARTP instrument, it is neces...

Embodiment 3

[0057] Example 3: 18S rDNA molecular identification

[0058] (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 -24 homogeneous disruptor to crush the cells, set the speed to 6.0 and the time to 40s; centrifuge the Lysing Matrix E Tube at 12000rpm for 10min to remove the sample debris; transfer the supernatant to a new EP tube and add 250μL of PPS reagent , shake the centrifuge tube 10 times to mix well, and 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, and let it stand for 3min to allow the DNA to 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-mi...

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Abstract

The invention relates to a fusarium fujikuroi mutant strain- fusarium fujikuroi GA-347 ( Fusarium fujikuroi GA-347 ) bred through an ARTP mutagenesis technique and an application of the fusarium fujikuroi mutant strain. The strain can be applied to industrial production in a large scale. The strain is preserved in China Center for Type Culture Collection, the address is Wuhan University, the postcode is 430072, the preservation No. is CCTCC NO:M 2019378, and the preservation date is 20 May 2019. In the growth process of the strain, conidium is not generated, the strain is inoculated to a liquid seed culture medium, and after cells grow to be mature, the cells are switched to a fermentation culture medium, so that the fermentation level is greatly increased, and the yield of gibberellin achieves 22 times of that of wild bacteria and leads in the national level. The gibberellin is in exocytosis, can be totally obtained from fermentation liquid, solid-liquid separation is facilitated, andthe extraction yield is increased. Besides, the utilization rate of equipment of raw materials is increased, the production cost is greatly reduced, and the strain has obvious economic benefits and social benefits.

Description

[0001] (1) Technical field [0002] The invention relates to a mutant strain of gibberella fujikura selected and bred by ARTP mutagenesis technology, and its application. [0003] (2) Background technology [0004] Gibberellins, as a class of natural plant growth regulators, have multiple biological functions on plant growth, can regulate plant rooting, germination, and fruiting processes, and can stimulate the growth and development of rice, cotton, and hemp crops. Therefore, it is widely used in hybrid rice seed production, citrus fruit preservation, cotton boll preservation, vegetable and young forest growth, and has great economic and social benefits for promoting agricultural production, especially grain production. In the beer industry, it can accelerate the germination of barley, make the germination of malt neat and uniform, and at the same time increase the activity of amylase, amylase and some proteases, thereby improving the quality of malt. The main active substanc...

Claims

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Application Information

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IPC IPC(8): C12N1/14C12N15/01C12N13/00C12P27/00C12R1/77
CPCC12N1/14C12N13/00C12N15/01C12P27/00C12N1/145C12R2001/77
Inventor 柳志强蒋欢张博郑裕国
Owner ZHEJIANG UNIV OF TECH
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