CN111154686B

CN111154686B - Natural ammonium-resistant nitrogen-fixing paenibacillus 23 and application thereof

Info

Publication number: CN111154686B
Application number: CN202010062921.6A
Authority: CN
Inventors: 陈三凤; 李琴
Original assignee: China Agricultural University
Current assignee: China Agricultural University
Priority date: 2020-01-19
Filing date: 2020-01-19
Publication date: 2021-08-10
Anticipated expiration: 2040-01-19
Also published as: CN111154686A

Abstract

The invention relates to the technical field of nitrogen-fixing microorganisms, and particularly relates to a natural ammonium-resistant paenibacillus azotobacter 23 and application thereof. The invention provides a Paenibacillus sp 23, which is preserved in China general microbiological culture Collection center with the preservation number of CGMCC No. 18767. The bacterium contains a structural gene nifH of azotase, can carry out high-efficiency biological nitrogen fixation under the condition of high-concentration ammonium (50-300mM), breaks through the inhibiting effect of the high-concentration ammonium condition on the biological nitrogen fixation, can be popularized and used in agricultural production, and effectively reduces the using amount of fertilizer.

Description

Natural ammonium-resistant nitrogen-fixing paenibacillus 23 and application thereof

Technical Field

The invention relates to the technical field of nitrogen-fixing microorganisms, and particularly relates to a natural ammonium-resistant paenibacillus azotobacter 23 and application thereof.

Background

Nitrogen is a major element essential for plant growth. The earth's surface is rich in nitrogen, but most of it exists in the atmosphere in the form of inert nitrogen gas and cannot be utilized by organisms. In order to ensure the high yield of grain crops, vegetables and fruits, a large amount of chemical nitrogen fertilizer is needed. However, the long-term excessive application of chemical nitrogen fertilizers can cause soil deterioration, environmental pollution and product quality reduction, and hinder the sustainable development of agricultural economy.

Biological nitrogen fixation refers to a process of reducing nitrogen in the air into ammonium by a few prokaryotic microorganisms under the action of in vivo nitrogen fixation enzymes. However, the biological nitrogen fixation efficiency is influenced by ammonium in the environment, and the high concentration of ammonium inhibits the nitrogen fixation effect. In other words, in poor soils, nitrogen fixing microorganisms can exert their nitrogen fixing effect sufficiently, whereas in fertile soils, nitrogen fixing microorganisms grow but not fix nitrogen. Generally, nitrogen-fixing microorganisms express nitrogenase in the presence of excess ammonium only if the ammonia assimilation mechanism is modified by chemical or genetic engineering. Almost all natural nitrogen-fixing bacteria can only fix nitrogen under low ammonium conditions. The nitrogen fixing activity of the currently reported nitrogen fixing microorganisms is low under the condition of high-concentration ammonium. The microorganism capable of efficiently fixing nitrogen under the condition of high-concentration ammonium is obtained, so that the nitrogen-fixing microorganism can fully play a nitrogen-fixing role in poor and fertile soil, and has important application value for reducing the using amount of chemical fertilizers in agricultural production.

Disclosure of Invention

In order to solve the technical problems in the prior art, the invention aims to provide a natural ammonium-resistant paenibacillus azotobacter 23 and application thereof.

In order to achieve the purpose, the technical scheme of the invention is as follows:

the invention provides an ammonium-resistant nitrogen-fixing Paenibacillus sp 23 strain, which is obtained by separation from natural environment, is preserved in China general microbiological culture Collection center (CGMCC for short, address: No. 3 of West Lu No.1 of Suzuku district of sunward areas in Beijing, China academy of sciences, postal code 100101) in 2019, 10 months and 30 days, is named as Paenibacillus sp, and has the preservation number of CGMCC No. 18767.

The colony of the Paenibacillus sp 23 on the LD solid culture medium is circular, convex, smooth in surface and complete in edge, and the diameter is about 0.5-1.5 mm.

The Paenibacillus sp 23 provided by the invention contains a structural gene nifH, and the nucleotide sequence for coding the gene is shown in SEQ ID NO. 1.

The 16S rDNA gene sequence of the Paenibacillus sp 23 is shown in SEQ ID NO. 2.

The invention also provides a microbial inoculum containing the Paenibacillus sp 23.

In the invention, the microbial inoculum containing Paenibacillus sp 23 can be a liquid microbial inoculum or a solid microbial inoculum. The microbial inoculum containing the Paenibacillus sp 23 can be prepared by adding auxiliary materials allowed in the field of microbial preparation by adopting a conventional technical means.

The azotobacter activity of the Paenibacillus sp 23 is obviously higher than that of the Paenibacillus strain Paenibacillus DUrus ATCC35681 which is recognized in the field and has the highest azotobacter activity, so that the Paenibacillus sp 23 has higher application potential.

Experiments prove that the Paenibacillus sp 23 can tolerate 50 to 300mM of high concentration ammonium, NH in the environment₄ ⁺When the concentration of the azotobacter reaches 50-300mM, the azotobacter can still keep higher azotobacter activity and carry out high-efficiency biological nitrogen fixation.

The invention provides application of the Paenibacillus sp 23 or a microbial inoculum containing the Paenibacillus sp 23 in biological nitrogen fixation.

In one embodiment of the present invention, the biological nitrogen fixation is performed under a low ammonium concentration condition in which the ammonium ion concentration is 0 to 5 mM. Preferably, the biological nitrogen fixation is performed under the condition that the concentration of ammonium ions is 0-4 mM. More preferably, the concentration of ammonium ion is 0-1 mM.

As another embodiment of the present invention, the biological nitrogen fixation is biological nitrogen fixation under a high ammonium concentration condition that the ammonium ion concentration is more than 20 mM.

The ammonium ion concentration in the high ammonium concentration condition where the ammonium ion concentration is more than 20mM may be 30 to 450 mM. Preferably 50 to 400 mM. More preferably 50 to 300 mM.

The invention provides application of the Paenibacillus sp 23 or a microbial inoculum containing the Paenibacillus sp 23 in fertilizer preparation.

The invention provides a biological fertilizer which comprises the Paenibacillus sp 23 or a microbial inoculum comprising the Paenibacillus sp 23.

The invention provides application of the Paenibacillus sp 23 or a microbial inoculum containing the Paenibacillus sp 23 or the biological fertilizer in plant planting.

The invention provides application of the Paenibacillus sp 23 or a microbial inoculum containing the Paenibacillus sp 23 in breeding nitrogen-fixing microorganisms.

The application in breeding the nitrogen-fixing microorganisms can be breeding the nitrogen-fixing microorganisms by utilizing Paenibacillus sp 23 through breeding methods such as mutagenesis, genetic engineering transformation and the like.

The invention provides application of the paenibacillus 23 or a microbial inoculum containing the paenibacillus 23 in promoting plant growth or improving plant yield.

Preferably, the above application is the administration of Paenibacillus sp 23 to the plant.

The present invention also provides a method for promoting plant growth or increasing plant yield, comprising: promoting plant growth or increasing plant yield by applying the Paenibacillus sp 23 or a fungicide comprising the Paenibacillus sp 23 to the plant.

Preferably, the Paenibacillus sp 23 is applied by soaking seeds with a suspension of the Paenibacillus sp 23 or irrigating the plant with a suspension of the Paenibacillus sp 23; or applying a fertilizer to the plant in combination with a suspension of paenibacillus 23.

The invention has the beneficial effects that: the invention provides a natural ammonium-tolerant Paenibacillus sp 23, which has excellent nitrogen fixation activity under the condition of low-concentration ammonium, can keep higher nitrogen fixation activity under the condition of high-concentration ammonium, and can carry out high-efficiency biological nitrogen fixation (the nitrogen fixation activity of the strain under the ammonium-free condition is 6967.66nmol C)₂H₄Permg protein h, nitrogenase activity in 100mM ammonium enrichment of 4600.23nmol C₂H₄Permg protein h, while the prior art natural nitrogen-fixing bacteria can only be generally 0-5 mM NH₄ ⁺Has azotobacter activity in the range), breaks the inhibiting effect of high ammonium condition on biological nitrogen fixation, ensures that the azotobacter can fully play the nitrogen fixation effect in poor and fertile soil, and has wide application prospect in agricultural production.

Drawings

FIG. 1 shows the colony morphology of the strain Bacillus Paenibacillus sp 23 of example 1 of the present invention on nitrogen-free plates.

FIG. 2 is an electrophoretogram of amplified products of nifH and 16S rDNA of the strain Paenibacillus sp 23 in example 1 of the present invention;

FIG. 3 shows the nitrogenase activity of the strain Paenibacillus sp 23 of example 2 of the present invention under different ammonium concentrations.

Detailed Description

Preferred embodiments of the present invention will be described in detail with reference to the following examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention will become apparent to those skilled in the art without departing from the spirit and scope of this invention.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1 isolation, culture and characterization of Paenibacillus 23

1. Paenibacillus 23 isolation

Paenibacillus 23 was isolated from a sample of the rhizosphere soil of conifer alpina in Beijing area.

2. Cultivation of Paenibacillus 23

Preparation of a culture medium: nitrogen-free liquid medium (1L): 20g of sucrose; 12.06g K₂HPO₄；3.4g KH₂PO₄；0.2g MgSO₄·7H₂O；0.01g NaCl；0.01g FeCl₃；0.002g NaMoO₄·2H₂O; adding H₂And O is metered to 1L. Sterilizing at 115 deg.C for 30 min. LD medium (1L): 5g yeast extract, 10g peptone, 2.5g NaCl. Sterilizing at 121 deg.C for 20 min. The solid medium was prepared by adding 1.5g of agar per 100ml of liquid medium.

Paenibacillus 23 was inoculated into a solid or liquid medium and cultured in an incubator at 30 ℃ for 2 days. The colony morphology of Paenibacillus 23 on nitrogen-free medium is shown in FIG. 1.

3. Identification of Paenibacillus 23

(1) Extraction of total DNA of bacteria: genomic DNA was extracted from a pure culture of the strain Paenibacillus 23 using a bacterial genome extraction kit (Tiangen corporation) according to the kit instructions.

(2) Identification of strains

Taking the bacterial genome DNA extracted in the step (1) as a template, and taking nifH F: GGCTGCGATCCVAAGGCCGAYTCVACCCG and nifH R: CTGVGCCTTGTTYTCGCGGATSGGCATGGC is a primer (wherein V represents A, G, C; Y represents C, T; and S represents G, C), the structural gene nifH of the azotobacter is amplified by PCR, and a band with the size of about 326bp is obtained. The band is recovered and purified, cloned to a pMD-18T vector, screened with blue white spots, picked with white spots and inoculated to a liquid culture medium containing ampicillin (Amp) resistance for overnight culture, and then quality-improved grains are sent to companies for sequencing after the plasmid enzyme cutting verification is correct. The nucleotide sequence of the gene nifH is shown in SEQ ID NO. 1. The sequencing result is compared with an online BLAST in a GenBank database, and the homology with the strain Paenibacillus sabinae T27 reaches 99.03 percent.

And (2) taking the bacterial genome DNA extracted in the step (1) as a template, designing primers (16S F: AGAGTTTGATCCTGGCTCAGAACGAACGCT and 16S R: TACGGCTACCTTGTTACGACTTCACCC) by using a 16S rDNA sequence of a model strain escherichia coli of prokaryotes, and carrying out PCR amplification on the 16S rDNA sequence. And (3) obtaining a band of about 1.5kb, recovering and purifying the band, cloning the band on a pMD-18T vector, screening blue white spots, picking white spots, inoculating the white spots into a liquid culture medium containing ampicillin (Amp) resistance for overnight culture, upgrading the grains, and sending the grains to a company for sequencing after the plasmid is cut and verified correctly. The 16S rDNA sequence is shown in SEQ ID NO.2, and the sequencing result is subjected to online BLAST comparison, so that the strain belongs to Paenibacillus (Paenibacillus).

The amplification system of the PCR is as follows: 10 × PCR buffer: 5 μ L, dNTP: 1.25 μ L, forward primer: 1.25 μ L, reverse primer: 1.25. mu.L, template DNA: 5 μ L, Taq DNA polymerase: 0.75. mu.L.

The amplification conditions for the above PCR were as follows:

amplification of nifH gene:

amplification of 16S rDNA:

the result of the electrophoresis detection of the PCR amplification product of the structural gene nifH of the azotobacter and the 16S rDNA is shown in figure 2.

Paenibacillus (Paenibacillus sp.)23 is deposited in China general microbiological culture Collection center (CGMCC for short, address: No. 3 of West Siro No.1 of Beijing Korean district, Microbiol institute of Chinese academy of sciences, postal code 100101) in 2019, 10.s and 30.s, and is classified and named as Paenibacillus sp, with the deposition number of CGMCC No. 18767.

Example 2 determination of the Nitrogen-fixing enzyme Activity of Paenibacillus 23 under different ammonium concentration conditions

1. Determination of nitrogenase Activity

The Paenibacillus 23 obtained in example 1 was inoculated into 5mL of LD medium, cultured overnight at 30 ℃, transferred into a 500mL triangular flask in an inoculum size of 1%, cultured at 30 ℃ for 8 hours, collected, and used in appropriate amounts of media with different ammonium concentrations (the media for measuring enzyme activity was supplemented with NH at different concentrations)₄Cl) suspension of the cells, adjustment of OD₆₀₀To 0.4. Inoculating 4mL of bacterial liquid into an anaerobic culture tube, pumping out air by using an air extractor, introducing argon, injecting 10% acetylene into the anaerobic tube, culturing at 30 ℃, and injecting 100 mu L of gas into a gas chromatograph every 2 hours to measure the ethylene content.

The minimal medium (1L) for measuring the enzyme activity is as follows: 26.3g Na₂HPO₄·12H₂O；3.4g KH₂PO₄(ii) a 10 μ g biotin; 26mg of CaCl₂·2H₂O；30mg MgSO₄；0.33mg MnSO₄·H₂O; 36mg ferric citrate; 7.6mg Na₂MoO₄·2H₂O; 10 μ g of p-aminobenzoic acid; 0.3g glutamic acid; 4g glucose. Sterilizing glutamic acid and glucose at 115 deg.C for 30min, adding into the mixture, and sterilizing other reagents at 121 deg.C for 20 min.

2. Method for determining protein content (Bradford,1976)

(1) Preparation of solutions

Bradford stock: 100mL of 95% ethanol, 200mL of 88% phosphoric acid, 350mg of Coomassie Brilliant blue G250;

bradford working solution: 425mL of double distilled water, 15mL of 95% ethanol, 30mL of 88% phosphoric acid, 30mL of Bradford stock; filter paper, store in brown bottle, and stand at room temperature. Can be stored for several weeks, but is filtered before use.

(2) Preparation of Standard Curve

1mL of Bradford working solution was put in a test tube, and 8. mu.L of 0.1mg/mL, 0.2mg/mL, 0.4mg/mL, 0.6mg/mL, 0.8mg/mL, and 1.0mg/mL BSA solutions were added, mixed well, left to stand for 3 to 5min, and developed into blue. Determination of OD₅₉₅The value is obtained. As a control, 1mL of Bradford working solution to which 8. mu.L of water was added was used. By OD₅₉₅Values are plotted on the ordinate and BSA solution concentration is plotted on the abscissa. The standard curve equation y is obtained as 0.6.5853x-0.1067, R²Is 0.9986.

(3) Protein content detection

And (3) centrifugally collecting thalli, adding 200 mu L of 0.5M NaOH, boiling for 5min, adding 200 mu L of HCl and 0.5M HCl, uniformly mixing, centrifugally absorbing 8 mu L of supernate, adding the supernate into 1mL of Bradford working solution, uniformly mixing, standing for 5min, and developing. Determination of OD₅₉₅The value is obtained. Will OD₅₉₅Values were taken into the standard curve equation and protein concentrations were calculated.

3. Calibration of 1nmol ethylene

(1) Preparing two 120mL serum bottles marked as 1# and 2# bottles;

(2) filling a serum bottle with water, plugging the serum bottle with a rubber plug inserted with a needle head to prevent bubbles from generating, taking down the rubber plug, pouring out 100mL of water (measured by a volumetric flask), and replacing with a new rubber plug, wherein the volume of air in the bottle is 100 mL;

(3) 2.24mL (2.24 mL in the standard case and the injection amount calculated from PV ═ nRT in the non-standard case) of ethylene was injected into the 1# bottle, 1mL of gas was taken out of the 1# bottle and injected into the 2# bottle, and 100. mu.L of gas was taken out of the 2# bottle and introduced into a gas chromatograph (model HP6890), and the peak area indicated was the amount of 1nmol of ethylene, from which it was possible to calculate how much nmol of ethylene was represented per unit peak area on the recording sheet.

(4) The calculation formula of the azotase activity is as follows:

the detection results of the azotobacter activity of the strain Paenibacillus 23 under different ammonium concentration conditions are shown in figure 3, the strain Paenibacillus 23 has the highest azotobacter activity under the ammonium-free (0mM) condition, and the azotobacter activity is 6967.66nmol C₂H₄/mg protein h; the azotase activity is very low under the condition of 5-20mM ammonium concentration; but the azotase activity is recovered with the continuous increase of the ammonium concentration, and the azotase activity is 4600.23nmol C under the condition of 100mM ammonium ion concentration₂H₄/mg protein h; and can still maintain higher nitrogen fixation activity under the condition of 200mM ammonium ion concentration, and the nitrogen fixation activity of the nitrogen fixation enzyme is 2605.24nmol C₂H₄/mg protein h; the azotase activity under the condition of 300mM ammonium ion concentration is 907.23nmol C₂H₄/mg protein h。

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Sequence listing

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Claims

1. An ammonium-tolerant nitrogen-fixing bacillus sp (Paenibacillus sp.)23 is preserved in China general microbiological culture Collection center with the preservation number of CGMCC 18767.

2. A microbial agent comprising the Paenibacillus sp 23 according to claim 1.

3. Use of the Paenibacillus sp 23 of claim 1 or the microbial inoculum of claim 2 in biological nitrogen fixation.

4. The use according to claim 3, wherein the biological nitrogen fixation is biological nitrogen fixation under the condition of low ammonium concentration with ammonium ion concentration of 0-5 mM.

5. The use according to claim 3, wherein the biological nitrogen fixation is biological nitrogen fixation at high ammonium concentrations greater than 20mM ammonium ion concentration.

6. The use according to claim 5, wherein the high ammonium concentration condition has an ammonium ion concentration of 30 to 450 mM.

7. The use according to claim 6, wherein the high ammonium concentration condition has an ammonium ion concentration of 50 to 400 mM.

8. Use of the Paenibacillus sp 23 of claim 1 or the microbial inoculum of claim 2 in the preparation of fertilizers.

9. A biofertilizer characterized by comprising the Paenibacillus sp (Paenibacillus sp.)23 according to claim 1 or the microbial agent according to claim 2.

10. Use of the Paenibacillus sp 23 of claim 1 or the microbial agent of claim 2 or the biological fertilizer of claim 9 in plant cultivation.

11. Use of the Paenibacillus sp 23 of claim 1 or the microbial inoculum of claim 2 for breeding nitrogen-fixing microorganisms.