CN108048384A - The nitrogen-fixing microorganism of resistance to ammonium and its construction method of a kind of glnA gene delections and application - Google Patents
The nitrogen-fixing microorganism of resistance to ammonium and its construction method of a kind of glnA gene delections and application Download PDFInfo
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- CN108048384A CN108048384A CN201711476764.8A CN201711476764A CN108048384A CN 108048384 A CN108048384 A CN 108048384A CN 201711476764 A CN201711476764 A CN 201711476764A CN 108048384 A CN108048384 A CN 108048384A
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- C12Y603/01—Acid-ammonia (or amine)ligases (amide synthases)(6.3.1)
- C12Y603/01002—Glutamate-ammonia ligase (6.3.1.2)
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Abstract
The present invention relates to microorganism field, a kind of nitrogen-fixing microorganism of resistance to ammonium of glnA gene delections and its construction method and application are specifically disclosed.The nitrogen-fixing microorganism of resistance to ammonium of the glnA gene delections is in no ammonium and has and can carry out biological nitrogen fixation under the conditions of ammonium, improve nitrogenase activity of the series bacillus microorganism belonging to genus under the conditions of high ammonium, high ammonium condition is breached to the inhibitory action of biological nitrogen fixation, is with a wide range of applications in agricultural production.
Description
Technical field
The present invention relates to microorganism field, specifically, be related to a kind of glnA gene delections the nitrogen-fixing microorganism of resistance to ammonium and
Its construction method and application.
Background technology
Nitrogen is required greatest member in plant production.Chemical nitrogen fertilizer ensure China's grain-production, vegetable cultivation and
It is played a very important role in cultivation of fruit tree.But it is long-term excessively to apply chemical nitrogen fertilizer partially, cause soil acidification and salination, soil
Microbial activity declines, oneself becomes an important restrictive factor of agricultural sustainable development.
Biological nitrogen fixation is a small number of bacteriums, and the nitrogen in air is reduced into the process of ammonium at normal temperatures and pressures, and fixed nitrogen is formed
Ammonium be absorbed and used by plants.But biological nitrogen fixation is a highly energy-consuming process, and enzyme reduces 1 dinitrogen, it is necessary to consume 16 ATP,
Therefore biological nitrogen fixation is by the stringent regulation and control of product ammonium.During ammonium concentration low (0-10mM), nitrogen-fixing bacteria carry out biological nitrogen fixation；Ammonium concentration is high
When (being more than 10mM), nitrogen-fixing bacteria are only grown without fixed nitrogen.That is, nitrogen-fixing bacteria nitrogen-fixing efficiency in dead soil is high,
Nitrogen-fixing efficiency is low in fertile soil.The inhibitory action of ammonium is broken through, is always a research hotspot in Biological Nitrogen Fixation Researches field.
The microorganism of the fixed nitrogen under the conditions of high ammonium is obtained, there is important application value in agricultural production.
The content of the invention
In order to solve the problems in the existing technology, the object of the present invention is to provide a kind of resistance to ammoniums of glnA gene delections
Nitrogen-fixing microorganism and its construction method and application.
In order to realize the object of the invention, technical scheme is as follows：
GlnA genes are almost present in all microorganisms.The product glutamine synthelase of glnA genes, the enzymatic
Glutamic acid and ammonium synthesis glutamine, glutamine provide ammonia for the synthesis of other amino acid.
First passage of the present invention has 2 glnA genes the study found that in fixed nitrogen series bacillus：1 glnA gene with
GlnR, which links together, forms glnRA transcriptional units, another glnA genes (being named as glnA1 genes here) individualism.
Under the conditions of high ammonium, the product glutamine synthelase of glnA genes (coming from glnRA transcriptional units) helps GlnR and fixed nitrogen base
Because the GlnR-binding site of the promoter region of cluster upstream are combined, inhibit the transcription of nif gene, and then inhibit fixed nitrogen enzyme activity
Property.GlnA gene delections or, relieve inhibitory action to nitrogenase activity.
Based on foregoing the study found that the present invention provides a kind of nitrogen-fixing microorganisms of resistance to ammonium of glnA gene delections, missing is such as
GlnA genes shown in SEQ ID NO.1.
Further, the microorganism is fixed nitrogen bacillus genus.
In the specific embodiment of the present invention, using Paenibacillus polymyxa as representing, to illustrate structure of the present invention
The mutant strain that method is built.
Further, the present invention provides institute by taking Paenibacillus polymyxa (Paenibacillus polymyxa) as an example
The construction method for the nitrogen-fixing microorganism of resistance to ammonium is stated, is included the following steps：
(1) it is homologous using primer up (glnRA)-F/up (glnA)-R amplification glnA upstreams shown in SEQ ID NO.2~3
Arm (1001bp), it is homologous using primer down (glnA)-F/down (glnA)-R amplifications downstream shown in SEQ ID NO.4~5
Arm (511bp)；
(2) by carrier pRN5101 BamHI/SalI restriction enzyme enzymatic treatments, endonuclease bamhi is obtained；
(3) using Gibson assembly master mix (New England Biolabs) to obtained by step (1)
Endonuclease bamhi obtained by upstream homology arm, downstream homology arm and step (2) is assembled, the recombinant plasmid transformed fixed nitrogen after assembling
Paenibacillus polymyxa (Paenibacillus polymyxa)；
(4) using vector plasmid pRN5101 carry Erythromycinresistant screening single exchange strains, by be commissioned to train more support after,
Therefrom screening again no longer has the bacterial strain of Erythromycinresistant, and glnA bases after confirmation obtains homologous double-crossover are verified and be sequenced through PCR
Because of the mutant strain after missing.
Further, the present invention provides the glnA genes shown in SEQ ID NO.1 to improve the micro- life of bacillus genus
Application of the object in terms of nitrogenase activity under the conditions of high ammonium, by making the glnA gene delections in the microbe genome DNA
Or be not expressed, improve series bacillus microorganism belonging to genus nitrogenase activity under the conditions of high ammonium.
The series bacillus microorganism belonging to genus is preferably Paenibacillus polymyxa.
High ammonium condition of the present invention refers to NH in microorganism local environment4 +Concentration be 100~400mM.
The nitrogen-fixing microorganism of resistance to ammonium of glnA gene delections provided by the invention in no ammonium and has ammonium condition (0~400mM
NH4 +) under, it is respectively provided with nitrogenase activity.And wild-type strain is only in 0-10mM NH4 +In the range of just have nitrogenase activity.
The present invention relates to raw material or reagent be ordinary commercial products, the operation being related to is unless otherwise specified
This field routine operation.
For example, BamHI/SalI restriction enzymes of the present invention are purchased from New England Biolabs.Described
Vector plasmid pRN5101 is purchased from BioVector plasmid vector bacterium cell gene collections.The mostly viscous class gemma of the fixed nitrogen
Bacillus is this laboratory screening bacterial strain, it is possible to use Paenibacillus polymyxa purchased from Shanghai Ke Wei chemical technologies Co., Ltd into
Row substitutes.
On the basis of common knowledge of the art, above-mentioned each optimum condition, can be mutually combined, obtain specific embodiment party
Formula.
The beneficial effects of the present invention are：
The present invention provides a kind of fixed nitrogen of resistance to ammonium series bacillus, in no ammonium and have that can to carry out biology under the conditions of ammonium solid
Nitrogen improves nitrogenase activity, breaches inhibitory action of the high ammonium condition to biological nitrogen fixation, has in agricultural production extensive
Application prospect.
Specific embodiment
The preferred embodiment of the present invention is described in detail below in conjunction with embodiment.It is it will be appreciated that following real
Providing merely to play the purpose of explanation for example is applied, is not used to limit the scope of the present invention.The skill of this field
Art personnel can carry out various modifications and replace to the present invention in the case of without departing substantially from spirit of the invention and spirit.
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
Embodiment 1
1st, glnA mutant strains screening structure
The product of glnA genes is glutamine synthelase (GS).There are two in Paenibacillus polymyxa
GlnA genes, one of them is connected to form a transcript unit with glnR genes：GlnRA, another glnA1 gene are individually deposited
.
The present invention only lacks the glnA genes in glnRA transcript units.
Utilize primer up (glnRA)-F/up (glnA)-R amplification glnA upstream homology arms (1001bp) in table 1, primer
Down (glnA)-F/down (glnA)-R amplification downstream homology arms (511bp), carrier pRN5101 are restricted with BamHI/SalI
Inscribe enzymatic treatment, then using Gibson assembly master mix (New England Biolabs) to above three
DNA fragmentation is assembled, the recombinant plasmid transformed fixed nitrogen Paenibacillus polymyxa Paenibacillus polymyxa after assembling,
The Erythromycinresistant carried using vector plasmid pRN5101 screens single exchange strains, by after being commissioned to train more and supporting, then therefrom screening not
There is the bacterial strain of Erythromycinresistant again, verify and be sequenced through PCR and is prominent after glnA gene delections after confirmation obtains homologous double-crossover
Mutant.
Table 1 builds the primer of glnA gene deletion strains
2. nitrogenase activity determination
GlnA mutant strains (Δ glnA) and wild-type strain (WT) are cultivated respectively in limit ammonium culture medium (- N) and the training of high ammonium
It supports in base (+N).Limit ammonium culture medium (- N) is addition 2mM glutamic acid in minimal medium, and high ammonium culture medium (+N) is in limit ammonium training
It supports and 100mM NH is added in base4Cl.Cultivate 10 it is small when more than measure nitrogenase activity (acetylene reduction method).
Fixed nitrogen enzyme activity (nmol C2H4/ mg protein hr) calculation formula be：
Experimental result is shown in Table 2. wild-type strains (WT) under the conditions of high ammonium almost without nitrogenase activity, and Δ glnA dashes forward
Mutant is respectively provided with nitrogenase activity under the conditions of limit ammonium and high ammonium.
2 nitrogenase activity of table compares
It should be appreciated that after the dosage of above-described embodiment agents useful for same or raw material is carried out equal proportion expansion or is reduced
Technical solution, it is substantially identical with above-described embodiment.
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Sequence table
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ggtatgcact gcaatcaatc gttgtttaag gacaatgaaa acgtatttta tgatgaaacg 780
gacgaactcg gattaagcca gacggcccgc cactatatgg ctggtattct gaaacatgca 840
cgtgcaatgg cagcgatcac gaatccaaca gtaaattcct acaaacgtct cgtacctggt 900
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Claims (7)
- A kind of 1. nitrogen-fixing microorganism of resistance to ammonium of glnA gene delections, which is characterized in that glnA of the missing as shown in SEQ ID NO.1 Gene.
- 2. the nitrogen-fixing microorganism of resistance to ammonium according to claim 1, which is characterized in that the microorganism is bacillus genus.
- 3. the nitrogen-fixing microorganism of resistance to ammonium according to claim 2, which is characterized in that the microorganism is mostly viscous class gemma bar Bacterium.
- 4. the construction method of the nitrogen-fixing microorganism of resistance to ammonium described in claim 3, which is characterized in that include the following steps：(1) primer up (glnRA)-F/up (glnA)-R amplification glnA upstreams homology arm shown in SEQ ID NO.2~3 is utilized (1001bp) expands downstream homology arm using primer down (glnA)-F/down (glnA)-R shown in SEQ ID NO.4~5 (511bp)；(2) by carrier pRN5101 BamHI/SalI restriction enzyme enzymatic treatments, endonuclease bamhi is obtained；(3) using Gibson assembly master mix (New England Biolabs) to the upstream obtained by step (1) Endonuclease bamhi obtained by homology arm, downstream homology arm and step (2) is assembled, and the recombinant plasmid transformed fixed nitrogen after assembling is mostly viscous Series bacillus Paenibacillus polymyxa；(4) using vector plasmid pRN5101 carry Erythromycinresistant screening single exchange strains, by be commissioned to train more support after, then from Middle screening no longer has the bacterial strain of Erythromycinresistant, and verifying and be sequenced glnA genes after confirmation obtains homologous double-crossover through PCR lacks Mutant strain after mistake.
- GlnA genes shown in 5.SEQ ID NO.1 are improving series bacillus microorganism belonging to genus nitrogenase activity under the conditions of high ammonium The application of aspect, which is characterized in that by making the glnA gene delections in the microbe genome DNA or not being expressed, carry High series bacillus microorganism belonging to genus nitrogenase activity under the conditions of high ammonium.
- 6. application according to claim 5, which is characterized in that the series bacillus microorganism belonging to genus is mostly viscous class gemma bar Bacterium.
- 7. application according to claim 5 or 6, which is characterized in that the high ammonium condition refers to NH in microorganism local environment4 + Concentration be 100~400mM.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110747142A (en) * | 2019-11-12 | 2020-02-04 | 中国农业大学 | Natural ammonium-resistant nitrogen-fixing microorganism LQ3 and application thereof |
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| CN101993849A (en) * | 2009-08-21 | 2011-03-30 | 中国科学院上海生命科学研究院 | Biological hydrogen production genetic engineering bacteria for relieving ammonium repression and construction method thereof |
| CN103314101A (en) * | 2010-11-03 | 2013-09-18 | 加利福尼亚大学董事会 | Biofuel and chemical production by recombinant microorganisms via fermentation of proteinacious biomass |
| CN106811416A (en) * | 2017-02-24 | 2017-06-09 | 中国农业科学院植物保护研究所 | Applications of the hydrolase CwlC in the cracking of bacillus mother cell |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101993849A (en) * | 2009-08-21 | 2011-03-30 | 中国科学院上海生命科学研究院 | Biological hydrogen production genetic engineering bacteria for relieving ammonium repression and construction method thereof |
| CN103314101A (en) * | 2010-11-03 | 2013-09-18 | 加利福尼亚大学董事会 | Biofuel and chemical production by recombinant microorganisms via fermentation of proteinacious biomass |
| CN106811416A (en) * | 2017-02-24 | 2017-06-09 | 中国农业科学院植物保护研究所 | Applications of the hydrolase CwlC in the cracking of bacillus mother cell |
Non-Patent Citations (1)
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110747142A (en) * | 2019-11-12 | 2020-02-04 | 中国农业大学 | Natural ammonium-resistant nitrogen-fixing microorganism LQ3 and application thereof |
| CN110747142B (en) * | 2019-11-12 | 2021-02-05 | 中国农业大学 | Natural ammonium-resistant nitrogen-fixing microorganism LQ3 and application thereof |
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