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Azotobacter chroococcum mutant with good nitrogen fixation capability and application thereof
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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: 2020-12-04
SHANGHAI ACAD OF AGRI SCI
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[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 mutationlibrary is obtained. The large-capacity mutationlibrary 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.
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Embodiment 1
[0044] The breeding method of the nitrogen-fixing bacterium sphaeroides mutant with good nitrogen-fixing ability of the present invention comprises the following steps:
[0045] 1) Prepare bacterial suspension
[0046] Inoculate the seed liquid of Azotobacter sphaeroides CICC21686 into the seed medium of Azotobacter phusticus, and incubate at 28 °C, 200 r min -1 Cultivate for 48 hours, take the bacterial solution into a sterile EP tube, and put it at 4000r min -1 , Centrifuge at 4°C for 10 min, discard the supernatant, suspend the cells with sterile saline, dilute to OD600 of 0.6-0.8, and obtain the cell suspension;
[0047] Azotobacter 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;
[0048] 2) ARTP mutagenesis
[0049] Mix the above bacteria suspension with 10% glycerol at a ratio of 1:1, and evenly spread...
Embodiment 2
[0075] The influence of embodiment 2 mutagenic nitrogen-fixing bacteria on plant growth
[0076] Pot experiments were used to inoculate nitrogen-free medium as a control treatment, inoculate Azotobacter brownus CICC21686, and inoculate mutant Azotobacter brownus A2820, a total of three treatments were used to test the effect of mutagenic nitrogen-fixing bacteria on the growth of maize.
[0077] The potting soil was taken from the agricultural experimental site of Shanghai Academy of Agricultural Sciences. Before the test, the soil was sieved with 2mm, sterilized at 121°C for 1 hour, and re-sterilized after cooling down. The potting container was 8cm×16cm×8cm (height × pot mouth diameter × pot bottom diameter), each pot is filled with the same amount of dry soil.
[0078] Treatment method: treat corn seeds with 95% alcohol for 5 minutes, pour off the alcohol, add 3% NaClO solution to treat for 2 minutes, pour off the NaClO solution, rinse with sterile water 5 times; sow 5 corn ...
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technical field [0001] The invention belongs to the field of microbes, and in particular relates to a mutant of Azotobacter sphaeroides with good nitrogen-fixing ability and an application thereof. Background technique [0002] As the world's largest producer and consumer of nitrogen fertilizer, my country's nitrogen fertilizer surplus in the agricultural system has become an environmental pollution factor. At present, the amount of nitrogen fertilizer used in China far exceeds the maximum crop yield demand. These excessive nitrogen fertilizer consumption has become the main factor of environmental pollution. . [0003] Biological nitrogen fixation is one of the most effective substitutes for industrial nitrogen fertilizers. By controlling the use of nitrogen fertilizers, it can be converted from traditional industrial nitrogen fertilizers to biological nitrogen fixation, improve the utilization rate of nitrogen fertilizers, and build an environment-friendly agriculture. At t...
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