A kind of nitrogen-fixing bacterium mutant with good nitrogen-fixing ability and its application

A technology of nitrogen-fixing bacteria and mutants of brown ball, applied in the field of microorganisms to achieve the effect of improving nitrogen-fixing efficiency

Active Publication Date: 2022-07-08
SHANGHAI ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] At present, ARTP has been successfully applied to the mutation breeding of more than 40 kinds of microorganisms including bacteria, fungi and microalgae. Compared with the traditional mutagenesis method, the chemically active particles in the jet can cause a very rich variety of DNA damage. Finally, a large-capacity mutation library is obtained. The large-capacity mutation library is easy to quickly obtain mutagenic strains with excellent performance and genetic stability, and the ARTP mutagenesis breeding technology is easy to operate, safe and non-toxic. At present, there are few reports on the mutagenesis of nitrogen-fixing strains.

Method used

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  • A kind of nitrogen-fixing bacterium mutant with good nitrogen-fixing ability and its application
  • A kind of nitrogen-fixing bacterium mutant with good nitrogen-fixing ability and its application
  • A kind of nitrogen-fixing bacterium mutant with good nitrogen-fixing ability and its application

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Experimental program
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Effect test

Embodiment 1

[0045] The method for selecting and breeding a nitrogen-fixing bacterium mutant with good nitrogen-fixing ability according to the present invention comprises the following steps:

[0046] 1) Preparation of bacterial suspension

[0047] Inoculate the seed liquid of Nitrogenococcus sphaeroides CICC21686 into the nitrogen-fixing bacteria seed medium at 28°C, 200 r min. -1 Incubate for 48h, take the bacterial solution into a sterile EP tube, at 4000r·min -1 , Centrifuge at 4°C for 10 min, discard the supernatant, suspend the cells with sterile saline, and dilute to an OD600 of 0.6-0.8 to obtain a cell suspension;

[0048] Nitrogen-fixing bacteria seed medium: yeast extract 0.5g, mannitol 20.0g, KH 2 PO4 0.2g, K 2 HPO 4 0.8g, MgSO 4 ·7H 2 O 0.2g, CaSO 4 ·2H 2 O 0.1g, FeCl 3 0.02g, Na 2 MoO 4 ·2H 2 O 0.03g, agar 15.0g, distilled water to 1.0L, pH7.2;

[0049] 2) ARTP mutagenesis

[0050] Mix the above-mentioned bacterial suspension with 10% glycerol at a ratio of 1:...

Embodiment 2

[0077] Example 2 The effect of mutagenic nitrogen-fixing bacteria on plant growth

[0078] A pot experiment was used to set up three treatments including inoculation of nitrogen-free medium as a control treatment, inoculation of Nitrogenobacter sphaeroides CICC21686, and inoculation of mutant A2820, to test the effect of mutagenic nitrogen-fixing bacteria on maize growth.

[0079] The potted soil was taken from the agricultural experimental site of the Shanghai Academy of Agricultural Sciences. Before the test, the soil was sieved with 2 mm, sterilized at 121 °C for 1 h, and then re-sterilized after cooling, and repeated 3 times. The potted container was 8 cm × 16 cm × 8 cm (height × The diameter of the pot mouth × the diameter of the pot bottom), and each pot is filled with an equal amount of dry soil.

[0080] Treatment method: treat corn seeds with 95% alcohol for 5 minutes, pour off the alcohol, add 3% NaClO solution for 2 minutes, pour off the NaClO solution, and rinse 5 ...

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Abstract

A kind of Azotobacter chroococcum mutant with good nitrogen-fixing ability and application thereof, it is Azotobacter chroococcum A2820, preservation number is CGMCC No.20443, preservation number is CGMCC No.20443, preservation date is 2020 On July 24th, the preservation unit is the General Microbiology Center of China Microorganism Culture Collection Management Committee, and its nitrogenase activity is 210-280 nmol·mg ‑1 ·h ‑1 The mutant of the present invention is a mutant with high enzymatic activity, which is mutagenized by ARTP for 28 seconds, measured and screened by acetylene reduction method, and has good nitrogen-fixing ability, and its genetic stability is confirmed by multiple subcultures.

Description

technical field [0001] The invention belongs to the field of microorganisms, and in particular relates to a nitrogen-fixing bacterium mutant with good nitrogen-fixing ability and application thereof. Background technique [0002] As the world's largest nitrogen fertilizer producer and consumer, China's nitrogen fertilizer surplus in the agricultural system has become an environmental pollution factor. At present, my country's nitrogen fertilizer use far exceeds the demand for the highest crop yield. These excessive nitrogen fertilizer consumption has become a major factor in environmental pollution. . [0003] Biological nitrogen fixation is one of the most effective alternatives to industrial nitrogen fertilizers. By controlling the use of nitrogen fertilizers, it can be converted from traditional industrial nitrogen fertilizers to biological nitrogen fixation, so as to improve the utilization rate of nitrogen fertilizers, build environment-friendly agriculture, and reduce g...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20A01H1/00C12R1/065
CPCC12N1/20A01H1/00C12R2001/065C12N1/205Y02P60/21
Inventor 黄艳娜唐雪明段赛菲王金斌宋丽莉
Owner SHANGHAI ACAD OF AGRI SCI
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