Gene capable of keeping efficient nitrogen fixing capability of strain

A nitrogen fixation and gene technology, applied in the field of genetic engineering, can solve the problems of no microbial start codon and stop codon, and no transcription and translation of proteins have been found, so as to achieve the effect of high nitrogen fixation activity

Active Publication Date: 2015-08-26
北京绿氮生物科技有限公司
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AI Technical Summary

Problems solved by technology

[0004] In the non-coding RNA ArsZ sequence (RNA sequence is SEQ ID NO: 1) of nitrogen-fixing Pseudomonas stutzeri, it is encoded by the corresponding arsZ gene (DNA sequence is SEQ ID NO: 2), and the gene coding reg

Method used

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  • Gene capable of keeping efficient nitrogen fixing capability of strain
  • Gene capable of keeping efficient nitrogen fixing capability of strain
  • Gene capable of keeping efficient nitrogen fixing capability of strain

Examples

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Effect test

Embodiment 1

[0054] Example 1 Functional verification of arsZ gene expression specifically responding to environmental nitrogen signals

[0055] The experimental steps are as follows:

[0056] (1) Activate Pseudomonas stutzeri A1501 bacteria in LB liquid medium, and culture overnight at 30°C;

[0057] (2) Centrifuge the thallus at 4000rpm / 10min the next day, and wash the thallus twice with normal saline;

[0058] (3) Suspend the bacteria with physiological saline and adjust the OD 600 ≈1.0;

[0059] (4) Cultivate the bacteria in the following phases: nitrogen and aerobic conditions (CNO, K medium), nitrogen-free and aerobic conditions (CN - No nitrogen source was added to the O, K medium, and argon was filled to exhaust the air for three minutes, and then 0.5% oxygen was injected), and the OD was adjusted 600 ≈0.5;

[0060] (5) After shaking the culture solution at 30°C for 0.5h, centrifuge at 8000rpm for 5min to collect the bacteria;

[0061] (6) Total bacterial RNA was extracted us...

Embodiment 2

[0067] Example 2 Nitrogenase activity comparison of nitrogen-fixing Pseudomonas stutzeri wild-type strain, arsZ deletion mutant strain and complementing bacterial strain

[0068] 1. Construction of arsZ gene deletion mutant strain in nitrogen-fixing Pseudomonas stutzeri A1501 (P. stutzeri A1501):

[0069] First, the upstream homologous fragment of the target gene, the chloramphenicol resistance box gene, and the downstream homologous fragment of the target gene were fused into a fusion fragment with a size of about 2.1 kb by fusion PCR technology, and then the cloned fragment was subjected to BamH I and Hind III After double enzyme digestion, it was connected to the suicide vector pk18mobsacB. The constructed suicide recombinant plasmid was introduced into the wild-type A1501 bacteria through the method of triparental combination, and the suicide plasmid was integrated into the chromosome through homologous recombination with the gene on the chromosome, and the single-crossove...

Embodiment 4

[0091] Determination of half-life of nitrogenase coding gene nifD mRNA in wild-type and arsZ deletion mutants in embodiment 4

[0092] The experimental steps are as follows:

[0093] (1) Pick a single colony from the plate and inoculate it into fresh LB liquid medium containing corresponding antibiotics, culture at 30° C., 220 rpm, on a shaker overnight.

[0094] (2) Transfer the bacterial solution to fresh nitrogen-free K medium for measuring nitrogenase activity, so that the initial OD 600 is 0.3.

[0095] (3) After 6 hours of induction culture under nitrogen fixation conditions, the nitrogenase activity value was measured and recorded, and at the same time, rifampicin with a final concentration of 200 μg / ml was added to the bacterial solution to inhibit RNA synthesis. After rifampicin was treated for 0, 1, 3, 5, and 7 minutes, centrifuge rapidly at 12,000 g for 2 minutes to remove the supernatant.

[0096] (4) Add 2 times the volume of rifampicin RNA later (Sigma Company...

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Abstract

The invention discovers a transcriptional functional non-coding RNA gene having the capability of keeping nitrogen fixing activity of a strain for the first time. The invention discovers that the gene can specifically respond to an external nitrogen signal, participate in the nitrogen fixing enzymatic activity regulation of nitrogen fixing pseudomonas stutzeri and keep the efficient nitrogen fixing capability of the strain, thereby being applicable to obtaining a gene engineering strain having efficient nitrogen fixing capability.

Description

Technical field: [0001] The invention belongs to the technical field of genetic engineering, and specifically relates to a gene capable of maintaining high-efficiency nitrogen fixation ability of bacterial strains and its application. Background technique: [0002] Bacterial non-coding RNA is usually located in the protein-coding intergenic region, usually 40-500 nucleotides in size, and can transcribe the RNA sequence, but not translate it into protein. In recent years, studies have found that bacterial non-coding RNA plays an important regulatory role in many life activities of prokaryotes, such as environmental stress, substance metabolism, quorum sensing, and bacterial virulence. [0003] In nitrogen-fixing bacteria, an increasing number of non-coding RNAs have been identified and studied. The functions of these elucidated small RNAs mostly focus on the conserved housekeeping function, the response of nitrogen-fixing bacteria to environmental signals, the resistance to ...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/70C12N1/21
Inventor 燕永亮林敏邓志平战嵛华陆伟
Owner 北京绿氮生物科技有限公司
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