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High-efficient solid nitrogen engineering bacteria, its construction and use thereof

A technology of engineering bacteria and nitrogen-fixing bacteria, which is applied in the direction of bacteria, the use of carriers to introduce foreign genetic material, organic fertilizers, etc., can solve the problems such as the limitation of bacterial energy supply, and achieve the effect of convenient screening

Inactive Publication Date: 2006-03-15
THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The purpose of the present invention is to introduce an extra copy of the dct gene into the combined nitrogen-fixing bacteria to solve the problem of the energy supply limitation of the bacteria, thereby obtaining a genetically engineered bacteria with high efficiency nitrogen fixation, and using the engineering bacteria to produce high-efficiency nitrogen fixation bacterial fertilizer

Method used

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  • High-efficient solid nitrogen engineering bacteria, its construction and use thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] The construction of embodiment 1 efficient Pseudomonas stutzeri

[0047] A. Alkaline lysis method to extract the plasmid DNA of C-1

[0048] 1) Inoculate a single colony into 5ml of liquid LB medium, shake at 200rpm at 37°C overnight.

[0049] 2) Take 1.5ml of the culture solution in an Eppendorf centrifuge tube and centrifuge at 12,000rpm for 2min.

[0050] 3) Discard the supernatant completely, suspend and precipitate the bacterial cells with 100 μl solution I (50 mmol / L sucrose, 10 mmol / L EDTA pH 8.0, 25 mmol / L Tris-HCl pH 8.0), and place on ice for 5 min.

[0051] 4) Add 200 μl of solution II (1% SDS, 0.2 mol / L NaOH), mix well by inverting up and down several times, and place on ice for 5 minutes.

[0052] 5) Add 150 μl solution III (5mol / L KAc+11.5ml glacial acetic acid, add water to 100ml), turn it upside down several times, and place it on ice for 5 minutes.

[0053] 6) Add 500 μl of phenol / chloroform / isoamyl alcohol (25:24:1), invert several times and mix well....

Embodiment 2

[0088] Example 2 Strain Nitrogen Fixation Activity Determination

[0089] It was determined by the acetylene reduction method (ARA).

[0090] ARA was determined by SP-2305 gas chromatograph.

[0091] 1) Pick a single colony and place it in LB liquid medium containing corresponding antibiotics for overnight culture.

[0092] 2) Take an appropriate amount of bacterial liquid and centrifuge, wash twice with sterile water, and resuspend the bacterial cells with sterile water to make the bacterial liquid OD 600 The value is about 0.6.

[0093] 3) Inoculate 10 ul of the bacterial liquid into a 7 ml penicillin vial containing 3 ml of nitrogen-free A15 semi-solid medium, and add a rubber stopper.

[0094] 4) After static cultivation at 30° C. for 24 hours, inject acetylene gas with a volume of 10% of the gas in the bottle, and continue the cultivation.

[0095] 5) The acetylene reduction activity was measured at 24 hours of incubation time.

[0096] The acetylene reducing activit...

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Abstract

A high-efficient nitrogen-fixing engineering bacterium, its construction and use are disclosed. The engineering bacterium is prepared by inducing copied plasmid containing DctBDP fragment with tetrodicarboxylic acid trans-gene into combined nitrogen-fixing engineering bacterium and obtaining high-efficient nitrogen-fixing engineering bacterium. It can be used for high-efficient nitrogen-fixing bacterial manure.

Description

Technical field: [0001] The present invention relates to a high-efficiency nitrogen-fixing engineering bacterium, especially a high-efficiency nitrogen-fixing engineering bacterium of Pseudomonas stutzeri constructed by using a four-carbon dicarboxylic acid transfer gene; the invention also relates to the construction of the engineering bacterium, and the production of bacteria using the engineering bacterium The use of fat. Background technique: [0002] Associative nitrogen-fixing bacteria are widely distributed in the rhizosphere of food crops such as rice, wheat, corn, and various fruit and vegetable plants. As a biological fertilizer source, it has many important biological characteristics such as nitrogen fixation and secretion of plant hormones. Combined nitrogen-fixing bacteria mainly bind to the root surface of plants and do not form specialized structures similar to root nodules. Compared with symbiotic nitrogen-fixing systems, the efficiency of combined nitrogen-f...

Claims

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Application Information

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IPC IPC(8): C12N15/63C12N1/20C12N15/70C05F11/08
Inventor 平淑珍李红权林敏陆伟张维徐玉泉陈明王丽英杨静李丹花
Owner THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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