Strain HB161398 with nitrogen fixation activity and application thereof

A technology of HB161398 and nitrogen fixation, applied in the field of genetic engineering, can solve problems such as the weakness of non-symbiotic nitrogen-fixing microorganisms, and achieve the effect of wide application range and good nitrogen fixation activity

Active Publication Date: 2019-05-14
海南热大一元和生态科技开发有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the research on non-symbiotic nitrogen-fixing microorganisms is generally weak, especially the mining and application of excellent strain resources

Method used

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  • Strain HB161398 with nitrogen fixation activity and application thereof
  • Strain HB161398 with nitrogen fixation activity and application thereof
  • Strain HB161398 with nitrogen fixation activity and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0014] The isolation of embodiment 1 bacterial strain HB161398

[0015] Mix the collected mangrove bottom mud (mangrove bottom mud at Qinglan Port, Wenchang, Hainan Province) and weigh 10g in a 100mL container. Put it in a Erlenmeyer flask with nitrogen-free medium and place it on a shaker to vibrate at 200r / min for 30min to fully disperse the soil sample. Take out the sample and let it stand for enrichment for 5 days, then take the upper layer liquid for gradient dilution, take 10 -3 -10 -6 Dilution applied to On nitrogen-free medium plates, 2 replicates per gradient. Inverted culture at 28°C for 5-7d. The strains with large colonies and transparent viscous shape were picked and purified by streaking on the plate. After purification, the 16s rDNA and nifH (nitrogen fixation) gene sequences were amplified and identified, and the strains were numbered and stored on a slant at 4°C and a glycerol tube at -80°C. Strain morphology such as figure 1 As shown, the cells are o...

Embodiment 2

[0031] The cultivation of embodiment 2 bacterial strain HB161398

[0032] The strain HB161398 can be preserved or temporarily stored by slant culture, and the strain HB161398 can be activated or transferred by plate culture, as follows:

[0033] (1) Slope culture: From the purified plate, pick a single colony of the strain and culture it for 3-5 days. Medium: 10g of peptone, 3g of beef extract, 1000mL of 50% old sea water, pH7.2, and store it at 4 ℃ refrigerator storage;

[0034] (2) Plate culture: From the purified plate, pick a single colony of the strain and culture it for 3-5 days. Medium: 10g of peptone, 3g of beef extract, 1000mL of 50% old sea water, pH7.2, and store it at 4 ℃ refrigerator storage.

Embodiment 3

[0035] Example 3 Detection of Nitrogen Fixation Activity of Bacterial Strain HB161398

[0036] Inoculate 10 mL of the Dobereiner nitrogen-free liquid medium with the pure bacterial strains preserved on the slant in Example 2, culture in a shaking table at 30°C for 72 hours, measure the OD600, and make uniform adjustments. Take 1 mL of the bacteria in sterilized 5 mL vials and cover with rubber stoppers. . The rim of the cap is sealed with paraffin. Inject 1 mL of acetylene with a syringe and incubate for 24 hours under the same conditions, then take 40 μL of the reacted gas, and use gas chromatography to measure the acetylene reducing activity (Acetylene reducing activity, ARA) of nitrogen-fixing bacteria.

[0037] The calculation method of acetylene reduction activity is as follows, wherein the ratio of the acetylene amount of acetylene reduction and fixed nitrogen adopts: C 2 h 4 :N 2 =4:1 for conversion):

[0038] ARA[nmol / (mL·h)]=Vst×Cst×Asa×Vtu / Vsa / Ast / h / 22.4

[003...

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Abstract

The invention discloses a strain HB161398 with the nitrogen fixation activity. The preservation number of the strain is CGMCC NO.13939, the preservation date is on Sep.18th, 2017, and the preservationunit is China General Microbiological Culture Collection Center (CGMCC for short), the preservation address is Microbiology Research Institute of China Science Academy, No.3, courtyard 1, western Beichen road, Chaoyang district of Beijing. The strain belongs to Rhodobacteraceae, and the nitrogen fixation activity is good. The invention further discloses application of the strain HB161398 for preparing fertilizer with the nitrogen fixation effect.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a bacterial strain HB161398 with nitrogen fixation activity. Background technique [0002] Nitrogen is the main element limiting ecosystem productivity, and biological nitrogen fixation is the main source of nitrogen in natural ecosystems. Biological nitrogen fixation includes three types: symbiotic, joint, and autogenous nitrogen fixation, among which joint nitrogen fixation and autogenous nitrogen fixation are collectively referred to as asymbiotic nitrogen fixation. Compared with symbiotic nitrogen fixation, although the rate of non-symbiotic nitrogen fixation is lower, it can survive and fix nitrogen without forming a symbiotic system with other organisms, and it is more widely distributed in time and space, and plays an important role in the nitrogen cycle of the ecosystem, especially the input of nutrients. contribute. As in many tropical forests, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C05G3/00C12R1/01
Inventor 刘敏童玉和李卫东
Owner 海南热大一元和生态科技开发有限公司
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