Endophytic azotobacter of wheat producing ACC (1-aminocyclopropane-1-carboxylate) deaminase and application thereof

A technology of deaminase and nitrogenase, applied in the application, bacteria, biocide and other directions, to achieve the effect of high nitrogenase activity and broad application prospects

Inactive Publication Date: 2011-11-23
INST OF AGRI RESOURCES & REGIONAL PLANNING CHINESE ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, the bottleneck restricting the development of my country'...

Method used

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  • Endophytic azotobacter of wheat producing ACC (1-aminocyclopropane-1-carboxylate) deaminase and application thereof
  • Endophytic azotobacter of wheat producing ACC (1-aminocyclopropane-1-carboxylate) deaminase and application thereof
  • Endophytic azotobacter of wheat producing ACC (1-aminocyclopropane-1-carboxylate) deaminase and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0044] Example 1, Isolation and Identification of Endophytic Azotobacter GDXM114 in Wheat

[0045] 1. Isolation of endophytic nitrogen-fixing bacteria in wheat

[0046] The specific operation for the isolation of wheat endogenous nitrogen-fixing bacteria is as follows: take fresh wheat plants (collected from Beijing, China), first rinse them with tap water, then soak them in 70% ethanol for 1 min, sterilize the surface with 2% sodium hypochlorite for 10 min, and aseptically clean them. Rinse with water 3 times. Under aseptic operation conditions, accurately weigh 10.0g of the sample, grind it into a paste in a sterile mortar, transfer and set the volume to 100ml, continue to dilute to make serial dilution samples, respectively from 10 -4 、10 -5 、10 -6 Take 0.1ml of the diluted solution and spread evenly on the above-mentioned CCM medium and nitrogen-free medium plate respectively, and invert culture at 28°C. After 3-4 days, pick a single colony and streak for purification t...

Embodiment 2

[0077] Example 2, Pseudomonas (Pseudomonas sp.) GDXM114 CGMCC No.5040 Nitrogenase Activity Determination

[0078] The Pseudomonas sp. (Pseudomonas sp.) GDXM114 CGMCC No.5040 that embodiment 1 is obtained carries out nitrogenase activity assay, and specific method is as follows: in 15 * 150mm screw-top glass tubes, add 5ml improved nitrogen fixation medium to make The slant was inoculated with nitrogen-fixing bacteria and cultured at 28°C. Azotobacter chroococcum (Azotobacter chroococcum) ACCC11103, which is commonly used in the production of microbial fertilizers, was used as the positive control, and the blank slant without inoculation was used as the negative control, with 3 replicates. After culturing for 72 hours, replace the rubber stopper, inject acetylene gas to make the final concentration 10%, seal it with medical adhesive tape, continue culturing for 72 hours, take 100 μl of reaction gas, measure the amount of ethylene produced by gas chromatography, and calculate th...

Embodiment 3

[0081] Example 3, Pseudomonas (Pseudomonas sp.) GDXM114 CGMCC No.50401-aminocyclopropane-1-carboxylic acid (ACC) deaminase activity assay

[0082] The Pseudomonas sp. (Pseudomonas sp.) GDXM114 CGMCC No.5040 that embodiment 1 is obtained carries out ACC deaminase activity assay, and specific method is as follows: take Azotobacter chroococcum (Azotobacter chroococcum) ACCC11103 as control group, carry out The obtained Pseudomonas (Pseudomonas sp.) GDXM114 CGMCC No.5040 screened in Example 1 was used as the experimental group. Use 5ml of nitrogen-free liquid medium to activate the strain, absorb 0.5ml of the culture medium and inoculate it into 60ml of the culture medium, incubate at 30°C for 24-48h, centrifuge at 4°C and 8000rpm for 10min to collect the bacteria, and use 15ml without (NH 4 ) 2 SO 4 The DF liquid medium was centrifuged to wash the cells twice, the cells were resuspended in 24ml ADF medium, cultured at 30°C for 24 hours, collected and the weight of the cells was...

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Abstract

The invention discloses endophytic azotobacter of wheat producing ACC (1-aminocyclopropane-1-carboxylate) deaminase and application thereof. The endophytic azotobacter of wheat is pseudomonas sp. GDXM114, and is collected in the China General Microbiological Culture Collection Center (CGMCC) with the collection number of CGMCC No.5040. The pseudomonas sp. GDXM114 with the collection number of CGMCC No.5040 has the advantages of higher nitrogenase activity, ACC deaminase activity and wide application prospect in producing nitrogenase microorganism inoculants and microbial organic fertilizers.

Description

technical field [0001] The invention relates to a wheat endophytic nitrogen-fixing bacterium producing ACC deaminase and its application. Background technique [0002] Nitrogen fertilizer is one of the most important means of production in agricultural production. The production of chemical nitrogen fertilizer consumes a large amount of energy, accounting for 70% to 80% of the total production cost. In recent years, energy has become increasingly tense, and the price of coal for chemical fertilizer production in my country has soared, leading to an increase in the price of chemical nitrogen fertilizers, which is unbearable for farmers and has a strong response. In addition, chemical nitrogen fertilizers have played a huge role in increasing crop yields and ensuring food security in my country, but unreasonable nitrogen application has formed serious non-point source pollution, causing a series of serious hazards, such as the shocking cyanobacteria incident in Lake Tai. Nit...

Claims

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Application Information

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IPC IPC(8): C12N1/20A01N63/00A01P21/00C12N9/02C12N9/88C05F11/08C05G3/00C12R1/38
Inventor 孙建光徐晶胡海燕高淼
Owner INST OF AGRI RESOURCES & REGIONAL PLANNING CHINESE ACADEMY OF AGRI SCI
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