Efficient artificial rhizosphere combined nitrogen fixation system

A nitrogen fixation and artificial technology, applied in the field of synthetic biology, can solve the problems of independence of microorganisms and plants, long research cycle, etc., and achieve the effect of overcoming long research cycle, good nitrogen fixation efficiency and growth promotion effect.

Active Publication Date: 2020-11-13
北京绿氮生物科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] However, the traditional method has many shortcomings such as long research cycle, ...

Method used

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  • Efficient artificial rhizosphere combined nitrogen fixation system
  • Efficient artificial rhizosphere combined nitrogen fixation system
  • Efficient artificial rhizosphere combined nitrogen fixation system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] The acquisition of embodiment 1Neb+amtR ammonium-secreting engineering bacteria

[0066] 1. Construction of amt gene deletion mutant strain in nitrogen-fixing Pseudomonas stutzeri A1501 (P. stutzeri A1501):

[0067] 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 4.1 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-crossover strain was obtained by resistance screening and PCR verification. Then, according...

Embodiment 2

[0069] Nitrogen fixation effect determination of embodiment 2Neb+amtR ammonium secreting engineering bacteria

[0070] 1. The purpose of the experiment: To investigate the nitrogenase activity and ammonium secretion ability of engineering bacteria (the growth curve, nitrogenase activity and ammonium secretion characteristics of nitrogen-fixing bacteria strains)

[0071] 2. Experimental object:

[0072] Control strain: wild-type Pseudomonas stutzeri A1501;

[0073] Nitrogen fixation gene mutant: nifH gene deletion mutant;

[0074] Ammonium-secreting engineering strain: Neb+amtR ammonium-secreting engineering strain;

[0075] 3. Experimental method:

[0076] Determination of enzyme activity of nitrogen-fixing bacteria strains, the method is as follows:

[0077] Pick the wild-type strains to be tested, ammonium-secreting engineering bacteria and nifH gene deletion mutant strains and inoculate them in LB liquid medium containing the corresponding antibiotics, and culture overn...

Embodiment 3

[0086] Example 3 Field application effect of the high-efficiency combined nitrogen fixation system constructed by ammonium-secreting engineering strains and corn

[0087] experimental method

[0088] (1) Determination of plant growth

[0089] The experiment is carried out in a smart greenhouse, which can control the temperature and humidity at all times. The nitrogen-fixing bacteria inoculated in the experiment included 5 treatments (ammonium-secreting engineering bacteria and wild-type strains, nifH gene deletion mutant strains, 50 mg N per kilogram of soil nitrogen treatment and no fertilization and no inoculation treatment control); corn varieties included 2 treatments (nitrogen high-efficiency utilization strains and control strains); the experiment set up 8 repetitions. The total setting of this experiment is 5×2×8=80 samples;

[0090] The corn seeds were soaked in sterile water for 30 minutes, then transferred to 5% NaClO solution for 1 minute, then treated in 75% eth...

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Abstract

The invention provides an efficient artificial rhizosphere combined nitrogen fixation system constructed according to synthetic biology theories and methods. Two brand-new functional modules are respectively and manually designed: a'Neb + amtR 'ammonium secreting gene module is constructed in a nitrogen-fixing microorganism chassis, a'Ham' nitrogen efficient utilization module is constructed in non-leguminous crop chassis of rice, corn, wheat and the like, and functional coupling of the two artificial modules is realized through inoculation technologies such as seed coating and the like at crop rhizosphere. Compared with a combined nitrogen fixation system in the prior art, the artificial rhizosphere combined nitrogen fixation system has higher biological nitrogen fixation efficiency and plant growth promoting effect.

Description

technical field [0001] The invention belongs to the technical field of synthetic biology, and specifically relates to a high-efficiency artificial rhizosphere combined nitrogen fixation system and its application. Background technique [0002] Nitrogen fertilizer input is a necessary way for agricultural producers to obtain high yields. However, nitrogen use efficiency is generally not high at present, leading to overuse. The plant rhizosphere is an active interface for soil-plant material exchange, and it is also the main activity area for the interaction between host plants and microorganisms. Combined nitrogen fixation systems widely exist in the rhizosphere of non-legume crops such as rice and corn, but the natural combined nitrogen fixation systems have many defects, such as being greatly affected by rhizosphere biological stress, low nitrogen fixation efficiency, and the stress resistance and tolerance of wild nitrogen-fixing mycorrhizal surfaces. The ammonium capaci...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/11C12N15/82A01H5/00A01H6/46C12R1/38
CPCC07K14/00C12N15/8243C12N15/74C07K14/415C12N15/8261C12N2510/00
Inventor 柯秀彬林敏燕永亮战嵛华陆伟
Owner 北京绿氮生物科技有限公司
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