CN115927234A - application of amt gene and mutant M107 in expression of azotobacter siderophin - Google Patents

Abstract

The invention relates to the technical field of bioengineering, in particular to application of an amt gene and a mutant M107 in expression of azotobacter siderophore. The mutant M107 with siderophin synthesis capacity obviously improved compared with that of a wild strain GXGL-4A is screened out by constructing a mutant library by utilizing a Tn5 transposition technology. The insertion site of mutant M107 is aminomethyltransferase gene amt. Through CAS plate detection and relative siderophore content measurement, the siderophore yield of the target mutant strain M107 is obviously improved compared with that of a wild strain, and the bacterial growth is not influenced; growth promotion experiments on cucumbers show that the obtained high-yield siderophin mutant strain has obvious growth promotion effect on the cucumbers. Compared with the prior art, the mutant strain provided by the invention has the advantages that the mutant strain can be used as a plant growth promoting strain, and the absorption of iron in soil is increased while the nitrogen fixation activity is maintained by mutating the amt gene of the azotobacter.

Description

application of amt gene and mutant M107 in expression of azotobacter siderophin

Technical Field

The invention relates to the technical field of bioengineering, in particular to application of an amt gene and a mutant strain M107 in expressing azotobacter siderophin.

Background

Iron is an essential element for microbial growth and is a component of a number of key enzymes that play important roles in electron transport, RNA synthesis, and resistance to reactive oxygen stress. The iron element is often Fe (OH) under physiological conditions ₃ The form (a) has poor iron solubility and cannot meet the requirement of microorganisms on iron. Under the low-iron environment, bacteria can synthesize para-Fe ³⁺ The high affinity, small molecular weight chelating factor (1-2 kDa), called siderophore, is secreted extracellularly or on the cell surface for Fe recovery ³⁺ And forms a siderophore-iron complex that transports iron from the extracellular to the intracellular compartment in association with the receptor protein on the cell membrane to meet the iron requirements for microbial growth. The secretion of siderophins is an important biocontrol mechanism for plant growth-promoting rhizobacteria. The growth-promoting bacteria can compete for limited iron in soil by producing siderophins, so that pathogenic bacteria can not grow or even die due to lack of iron, and the purpose of biological control is achieved. In recent years, scholars at home and abroad have made intensive studies on the biosynthesis of siderophore and the absorption and transport mechanism of siderophore for iron. Screening high-yield siderophore by improving culture conditions of bacteriaThe method improves the capability of microorganisms for producing the siderophore by using the biotin strain, the receptor for introducing the exogenous siderophore, siderophore biosynthesis regulation and the like, and achieves certain progress. However, the related studies are mostly targeted to escherichia coli and pseudomonas aeruginosa. The genetic engineering method is adopted to improve the biosynthesis capacity of the siderophin so as to further improve the biocontrol capacity of the growth-promoting bacteria or the biocontrol bacteria, and the method has wide application prospect. In addition, many researches prove that the siderophore plays an important role in promoting the growth and development of plants, improving the stress resistance of crops and the like.

The combined azotobacter has good biological nitrogen fixation capacity, and particularly, the genetic engineering development of the azotobacter derived from non-leguminous plants is a research hotspot at present. The azotobacter has the advantages of wide host range, convenient culture, wide source and the like, and if the azotobacter can be developed into a bacterial strain with high siderophin yield, the azotobacter can fully exert the azotobacter and iron absorption capacity of the bacteria, and improve the growth promotion and disease prevention capacity, thereby being beneficial to the growth and development of host plants. The biosynthesis of siderophore belongs to the non-ribosomal peptide synthesis pathway, and is characterized by that it uses a kind of multifunctional protein complex, i.e. non-ribosomal peptide synthetases (NRPS) to recognize, activate and transfer amino acid substrate and synthesize non-ribosomal peptide (NRPS) according to a specific sequence. Siderophins are important secondary metabolites produced by many bacteria and fungi, etc., and are synthesized by siderophin synthesis gene clusters. The complete siderophore synthesis gene cluster is analyzed by measuring the whole genome sequence of bacteria or fungi through the bioinformatics means, and the modification of the core siderophore synthesis enzyme gene is a way to improve the siderophore production capability of microorganisms. However, the biosynthesis of siderophore is influenced by many factors such as bacterial culture conditions, iron ion concentration, siderophore receptors, iron signal transduction and transport, in addition to gene regulation. Therefore, it is not really easy to improve the ability of microorganisms to produce siderophore by modifying part of the genes in the siderophore synthesis gene cluster. In order to better develop and utilize plant growth-promoting bacteria including azotobacter, the bacteria are developed into multifunctional biological agents, and a new idea is to try to improve the siderophilic capacity of the bacteria.

Disclosure of Invention

In order to solve the problems, the invention aims to provide the application of the amt gene and the mutant strain M107 in expressing the siderophore of azotobacter. The mutant M107 with siderophin synthesis capacity obviously improved compared with that of a wild strain GXGL-4A is screened out by constructing a mutant library by utilizing a Tn5 transposition technology. The insertion site of mutant M107 is aminomethyltransferase gene amt. Through CAS plate detection and relative content measurement of siderophins, the siderophin yield of the target mutant strain is confirmed to be remarkably improved compared with that of a wild strain, and bacterial growth is not influenced. Growth promotion experiments on cucumbers show that the obtained high-yield siderophin mutant strain has obvious growth promotion effect on the cucumbers. Compared with the prior art, the mutant strain provided by the invention has the advantages that the mutant strain can be used as a plant growth promoting strain, the absorption of iron in soil is increased while the nitrogen fixation activity is maintained, the plant root system can obtain more iron, and the plant biomass is improved. Meanwhile, the mutant strain inhibits the growth of soil pathogenic bacteria by competing the iron nutrition of the pathogenic bacteria in the soil, and is beneficial to improving the disease resistance of plants.

The purpose of the invention can be realized by the following technical scheme:

the first purpose of the invention is to provide an application of a target gene in expressing azotobacteria siderophin, wherein the target gene is an amt gene, and the DNA sequence of the amt gene is shown as SEQ ID NO. 1.

The second purpose of the invention is to provide the application of the mutant M107 in expressing azotobacter siderophin, wherein the preservation number of the mutant M107 is CGMCC No.24401.

In one embodiment of the invention, the method for applying the mutant M107 in expressing azotobacter siderophin to promote plant growth comprises the following steps:

(1) Inoculating the mutant M107 into an LB culture medium, and collecting a mutant M107 cell precipitate after overnight culture;

(2) And (2) treating the mutant strain M107 cell sediment collected in the step (1) and applying the treated mutant strain M107 cell sediment to the rhizosphere of the plant.

In one embodiment of the present invention, in step (1), the mutant M107 was inoculated into LB medium at an inoculum size of 1%.

In one embodiment of the present invention, in step (2), the post-treatment is to resuspend the mutant M107 cell pellet in sterile water and then centrifuge, and this process is repeated several times, and the mutant M107 cell pellet obtained by the last centrifugation is resuspended in sterile water to obtain a mutant M107 cell suspension.

In one embodiment of the invention, the mutant M107 cell suspension is applied while the plant is in the two-leaf one-heart stage.

In one embodiment of the invention, 5-10mL of mutant M107 cell suspension is released per plant with a bacteria count of 1X 10 ⁸ CFU/mL; the frequency was 1 application every 5 days with 5 consecutive treatments.

The third purpose of the invention is to provide the application of a substance for knocking out or replacing a target gene in the preparation of a high-yield siderophil azotobacter microbial inoculum or a siderophil growth-promoting microbial inoculum, wherein the target gene is an amt gene, and the DNA sequence of the amt gene is shown in SEQ ID NO. 1.

The fourth purpose of the invention is to provide the application of the mutant M107 in the preparation of high-yield azotobacter siderophilus or growth-promoting bacterium agent for producing siderophilus, wherein the preservation number of the mutant M107 is CGMCC No.24401.

The fifth purpose of the invention is to provide a mutant M107, wherein the preservation number of the mutant M107 is CGMCC No.24401.

The invention adopts a gene mutation method, and the improvement of the siderophin production capability of the strain is realized by the insertional inactivation of a target gene, thereby being a controllable and efficient way. The method is simple to use, the inactivation of the target gene is easy to control and realize, the method has universal applicability, and the method has good popularization value in the aspects of siderophin production regulation and control of plant growth promoting bacteria, azotobacter and the like.

Compared with the prior art, the invention has the following beneficial effects:

(1) According to the invention, the target gene amt is inactivated by insertion mutation, so that the siderophin biosynthesis is obviously improved, the operation is simple, the result is reliable, and the effect is obvious. The research finds that the gene is closely related to the synthesis yield of the siderophore and is presumed to be a negative regulation mechanism, but the specific regulation mechanism of the gene is not clear at present.

(2) The high expression of the siderophin in the invention can not cause any harm to bacterial cells, the growth and the propagation of bacteria are not influenced, and the morphological characteristics and the physiological and biochemical processes of the bacteria are not obviously changed. The obtained transformant can remarkably promote the growth of plants as a growth-promoting bacterium compared with a control wild strain.

(3) The invention firstly confirms that the amt gene can regulate the biosynthesis of the siderophore.

(4) The mutant strain created by the invention can be applied to the preparation of azotobacter inoculum for high yield of siderophin or growth-promoting inoculum for siderophin. The high-yield mutant strain of the siderophin constructed by genetic operations such as gene knockout and the like can be used as high-efficiency growth-promoting bacteria, particularly can remarkably improve the iron absorption of plants when soil is lack of iron or low in iron, and can be used in the field of agricultural production or ecological environment protection after being prepared into a microbial inoculum.

Drawings

FIG. 1 is a competent cell morphology of azotobacter GXGL-4A after hypertonic culture for transposition mutation; FIG. 1A: conventional LB culture; FIG. 1B: LB culture containing 15g/L NaCl; FIG. 1C ₂ Replace NaCl in regular LB medium).

FIG. 2 shows qualitative and quantitative determination of the siderophore biosynthetic ability of mutant M107.

FIG. 3 is a schematic diagram of the transposition insertion site of mutant M107.

FIG. 4 shows PCR cloning of the amt gene and identification of the transposition site of mutant M107; m: DNA molecular weight Marker;4A: amplifying a target gene amt by using the genomic DNA of a nitrogen-fixing bacteria wild strain GXGL-4A as a template; m107: and amplifying the mutated target gene amt by using the mutant M107 genome DNA as a template. .

FIG. 5 is a phylogenetic tree of the amt gene; wherein, E: escherichia; within parentheses are the strain genome accession numbers in GenBank.

FIG. 6 is fluorescent quantitative PCR of mutant M107 mutant site gene; the amt gene in GXGL-4A is relatively quantitative 1, and M107 indicates the relative expression levels of trX and amt, which are target genes after mutation. The fluorescent quantitative PCR experiment takes rpoB gene as an internal reference gene.

FIG. 7 is a growth curve of the target strain.

FIG. 8 shows the growth promotion effect of the target strain on cucumber, and FIG. 8A shows the effect of the mutant M107 treatment on the plant height of cucumber; FIG. 8B is the effect of mutant M107 treatment on cucumber root fresh weight; FIG. 8C is the effect of mutant M107 treatment on cucumber plant fresh weight; FIG. 8D is the effect of mutant M107 treatment on cucumber root length.

FIG. 9 is a pictorial view of the growth promoting effect of the objective strain on cucumber.

FIG. 10 is a test of the inhibition effect of target strains on the growth of corn northern leaf blight pathogenic bacteria.

Detailed Description

The invention provides an application of a target gene in expressing azotobacter siderophore, wherein the target gene is an amt gene, and the DNA sequence of the amt gene is shown as SEQ ID NO. 1.

The invention provides application of a mutant strain M107 in expressing azotobacter siderophore, wherein the preservation number of the mutant strain M107 is CGMCC No.24401.

In one embodiment of the invention, the application method of the mutant M107 in expressing azotobacteria siderophore for promoting plant growth comprises the following steps:

(1) Inoculating the mutant M107 into an LB culture medium, and collecting a mutant M107 cell precipitate after overnight culture;

(2) And (2) treating the mutant strain M107 cell sediment collected in the step (1) and applying the treated mutant strain M107 cell sediment to the rhizosphere of the plant.

In one embodiment of the present invention, in step (1), the mutant M107 was inoculated into LB medium at an inoculum size of 1%.

In one embodiment of the present invention, in step (2), the post-treatment is to resuspend the mutant M107 cell pellet in sterile water and then centrifuge, and this process is repeated several times, and the mutant M107 cell pellet obtained by the last centrifugation is resuspended in sterile water to obtain a mutant M107 cell suspension.

In one embodiment of the invention, the mutant M107 cell suspension is applied while the plant is in the two-leaf one-heart stage.

In one embodiment of the invention, 5-10mL of mutant M107 cell suspension is released per plant with a bacteria count of 1X 10 ⁸ CFU/mL; the frequency was 1 application every 5 days with 5 consecutive treatments.

The invention provides application of a substance for knocking out or replacing a target gene in preparation of a high-yield siderophin azotobacter microbial inoculum or a siderophin growth-promoting microbial inoculum, wherein the target gene is an amt gene, and the DNA sequence of the amt gene is shown as SEQ ID NO. 1.

The invention provides application of a mutant M107 in preparation of a high-yield siderophil azotobacter microbial inoculum or a siderophil growth-promoting microbial inoculum, wherein the preservation number of the mutant M107 is CGMCC No.24401.

The invention provides a mutant M107, wherein the preservation number of the mutant M107 is CGMCC No.24401.

The invention adopts a gene mutation method, and the improvement of the siderophore production ability of the strain through the insertional inactivation of the target gene is a controllable and efficient way. The method is simple to use, the inactivation of the target gene is easy to control and realize, the method has universal applicability, and the method has good popularization value in the aspects of siderophin production regulation and control of plant growth promoting bacteria, azotobacter and the like.

The invention is described in detail below with reference to the figures and specific embodiments.

In the following examples, materials used are commercially available unless otherwise specified; the technology or the detection means are the conventional technology or detection means in the field unless specified otherwise.

Example 1

This example provides a mutant M107.

(1) Preparation of azotobacter GXGL-4A competent cell

The azotobacter united GXGL-4A is separated from the root of maize in Guangxi Guilin, and the accession number of the whole genome sequence in GenBank is CP015113.1; the preservation number of GXGL-4A in China general microbiological culture Collection center (CGMCC) is CGMCC No.12588.

The method for preparing competent cells by GXGL-4A comprises the following steps:

1) Streaking on a common LB plate for culture, selecting a single colony, and inoculating the single colony into a conventional LB culture medium for overnight culture;

2) Inoculating with 1% inoculum size in CaCl containing 7.5g/L ₂ HO-2 medium (modified LB medium, no NaCl) to logarithmic phase;

3) And (4) centrifugally collecting bacterial precipitates at low temperature, and washing impurities and thallus fragments by using a PEB electric shock buffer solution to obtain bacterial competent cells.

(2) Morphological observation of azotobacteria GXGL-4A competent cells

Culturing azotobacter GXGL-4A in HO-2 solution to OD ₆₀₀ When the concentration reaches 0.6-0.8, 1mL of azotobacter GXGL-4A bacterial liquid is sucked and centrifuged for 5min at 8000rpm, the supernatant is discarded, the azotobacter GXGL-4A bacterial liquid is washed by equivalent sterile water, the operation is repeated twice, finally, the azotobacter GXGL-4A bacterial liquid is re-suspended by sterile water, 10 mu L of re-suspended bacteria is dripped on a copper sheet, and the copper sheet is dried in the shade at night. The cells were observed under a transmission electron microscope with the competent cells prepared by culturing in a conventional LB, HO-1 (LB medium containing 15 g/NaCl) medium as a control. The result shows that the capsule and cytoplasm of azotobacter GXGL-4A cultured by common LB are combined and compacted evenly, and no obvious limit is seen. The azotobacter GXGL-4A cultured by HO-1 shows the phenomenon of cytoplasmic shrinkage, the color of cytoplasm is dark, the thickness of the capsule is uneven, and the capsule and the cytoplasm have obvious limits. The shrinking of azotobacter GXGL-4A under the HO-2 culture condition is more serious, and the morphology is distorted. As a result, the morphology of the cells varied significantly between the different culture methods (FIG. 1).

(3) Electric shock transformation and Tn5 mutant strain screening

1) Tn5 transposition complex was added to a PEB solution containing azotobacter GXGL-4A competent cells to a final concentration of 2.5. Mu.g/mL, and the mixture was ice-cooled for 5 minutes. Then, 200. Mu.L of the mixed solution was added to a pre-cooled Bio-Rad cuvette at 0 ℃ with an electrode distance of 0.2cm, and the cuvette was shocked by a Bio-Rad MicroPulser shock converter at a voltage of 2.0Kv for a period of 2ms. After the completion of the electric shock, 800. Mu.L of SOC medium was added immediately, and the mixture was incubated at 37 ℃ and 150rpm for 1 hour.

2) Plate coating and transformant screening: 100 μ L of the recovered and grown cells were spread on a plate containing Km (50 μ g/mL) antibiotic, cultured at 37 ℃ for 16 hours in an incubator, and transformants were picked and identified after pure culture.

SOC recovery medium: contains 0.5% (W/V) yeast extract, 2% (W/V) tryptone, 10mmol/L NaCl, 2.5mmol/L KCl, 10mmol/L MgCl ₂ 、20mmol/L MgSO ₄ 20mmol/L glucose. Sterilized under 8 lbs pressure for 20min and stored at 4 deg.C until use.

PEB shock buffer: contains 272mmol/L sucrose and 1mmol/L MgCl ₂ 。

3) Identification of positive transformants: and (3) designing 4 pairs of primers by taking an anfD gene of azotobacter GXGL-4A and a gene fragment on a Tn5 transposable element as amplification target fragments. Wherein the primer pair anfD/anfR is used for amplification so as to ensure that a transformant is derived from azotobacter GXGL-4A; the remaining 3 pairs of primers, screen 1-F/Screen 1-R, screen-F/Screen 2-R and Screen 3-F/Screen 3-R, were designed based on the Tn5 gene sequence, and the insertion of Tn5 elements in the transformant genome, i.e., the transposition of the transformant, was ensured by amplifying the desired fragments of the 3 sets of primers. And performing PCR amplification by using the total DNA of the bacterial transformant as a template. PCR procedure: pre-denaturation at 94 deg.C for 10min, denaturation at 94 deg.C for 30s, annealing at 54 deg.C for 30s, and elongation at 72 deg.C for 1min, for 35 cycles, and final elongation at 72 deg.C for 7min. The sequences of the 4 pairs of primers are as follows:

anfD：5′-CGGGCAATCTCTTCATCAAT-3′(SEQ ID NO.2)，

anfR:5'-ATACCTTCGCGACCGATATG-3' (SEQ ID NO. 3), and the target product 655bp;

Screen 1-F：5′-CAGGGATCTGCCATTTCATT-3′(SEQ ID NO.4)，

screen 1-R:5'-GCCTGAGCGAGACGAAATAC-3' (SEQ ID NO. 5), target product 922bp;

Screen 2-F：5′-GGACGCGATGGATATGTTCT-3′(SEQ ID NO.6)，

screen 2-R:5'-GCCTGAGCGAGACGAAATAC-3' (SEQ ID NO. 7), the target product 602bp;

Screen 3-F：5′-ATTCAACGGGAAACGTCTTG-3′(SEQ ID NO.8)，

screen 3-R:5'-ATTCCGACTCGTCCAACATC-3' (SEQ ID NO. 9), target 656bp.

Example 2

This example provides a method for detecting the siderophore synthesis ability of azotobacter mutant strains.

(1) Preparation of CAS detection plate

1) 200mL of phosphate buffer solution preparation: 1.924g Na ₂ HPO ₄ 、0.9082g NaH ₂ PO ₄ 、0.15g KH ₂ PO ₄ 、0.5g NH ₄ Cl, 0.25g NaCl, pH adjusted to 6.8-7.0 with 1mol/L NaOH.

2) Preparation of CAS solid detection Medium: prepare A, B liquid separately.

Solution A: two beakers are taken and respectively weighed with 40mL of double distilled water, 50mL of double distilled water and 0.0605g of CAS-S and 0.0729g of CTAB (HTDMA). And (3) heating and dissolving CTAB until the CTAB is clear, pouring CAS-S into the CTAB solution, stirring while adding (the sequence of the steps cannot be reversed, otherwise, a large amount of precipitate may appear), and uniformly mixing at high temperature and high pressure for later use. Preparing 1mmol/L FeCl ₃ (dissolved in 10mmol/L HCl) and filter sterilized with a 0.22 μm microfiltration membrane (iron salt solution autoclaving ease). After CTAB-CAS cooling, 10mL FeCl was added ₃ The solution was shaken up to obtain 100mL of CAS stain with no precipitate.

And B, liquid B: 50mL of phosphate buffer solution, 60mL of acid hydrolyzed casein, 2mL1 mmol/L of CaCl ₂ ，2mL10mmol/L MgSO ₄ Adjusting the pH value to 6.9 by 1mol/L NaOH, and metering to 1L by 20g of agar.

And after sterilization, adding 10mL of CAS staining agent into 100mL of the B solution, shaking uniformly, measuring 30mL of CAS staining agent, pouring into a culture dish with the diameter of 9cm to obtain a blue CAS solid detection culture medium, and cooling and solidifying for later use.

(2) CAS plate screening for siderophore synthesis capacity

The petri dish with an inner diameter of 9cm was marked and divided into 6 regions, and 3 parallel samples of 3 GXGL-4A wild strains and 1 mutant strain were inoculated to the 6 regions, respectively. After 24h of culture, the sizes of yellow halos generated by the mutant strains and wild strains are observed, and mutant strains with obviously changed siderophin production capacity are screened out by comparing the sizes of the halos.

(3) Quantitative detection of siderophore synthesis

After preliminary screening by CAS plate detection method, the product is obtainedThe obtained target mutant strain is re-screened by a quantitative detection method. Selecting mutant strain and wild strain by inoculating loop, and culturing in liquid LB to OD ₆₀₀ The value is 0.7-0.8, 600 mu L of bacterial liquid is taken and put in a 1.5mL centrifuge tube, centrifuged for 5min at 8000rpm, and then supernatant is taken, and the volume ratio is 1:1 and CAS detection solution, standing for 1h after mixing uniformly, measuring the light absorption value (As) under the wavelength of 630nm, and taking double distilled water As a contrast for zero adjustment. Mixing blank culture medium with CAS detection solution, taking its light absorption value As reference value (Ar), and mixing according to [ (Ar-As)/Ar]X 100% the relative amount of siderophore produced by the mutant was calculated.

CAS quantitative assay solution (100 mL): the preparation step refers to the solution B, and the dosage of each reagent is as follows: 0.009g CAS-S,0.0218g CTAB,1.5mL 1mmol/L FeCl ₃ 。

Example 3

This example provides the identification of a highly siderophilic mutant M107.

(1) Quantitative and qualitative identification of high-yield siderophore

Through CAS flat plate multi-round primary screening and CAS detection liquid quantitative detection re-screening, mutant strains M107 (Kosakonia radicitans M107) with high yield of siderophins are finally screened from 1633 mutant strains, and the siderophin production capacity of the mutant strains is obviously improved compared with that of wild strains GXGL-4A (figure 2); the mutant M107 has been preserved for a long time in China general microbiological culture Collection center (CGMCC), and the preservation number is CGMCC No.24401.

(2) Determination of mutant M107 Gene

Sequencing a target mutant strain through a whole genome, and finally determining that the mutant strain is inserted in a single copy way, wherein the insertion sites are respectively in coding regions of an amt gene (the DNA sequence of which is shown in SEQ ID NO. 1) (arcus tag: A3780-01680) (the coding sequence of the gene is mutated from SEQ ID NO.1 to SEQ ID NO. 16) (figure 3). In order to further identify the obtained mutant strain as the single-copy transposition mutation of the target gene, forward and reverse primers are designed according to flanking sequences at two ends of the mutant gene obtained by genome sequencing for amplification, an amplification product obtained by taking the genome DNA of a wild strain GXGL-4A as a template is taken as a contrast, the amplification product of the mutant strain can be increased by 2019bp, and if the obtained result is consistent with the expectation, the mutant gene can be confirmed. The primer pairs used were as follows:

M107-amt-F：5′-cgcctataacgaaccgatgg-3′(SEQ ID NO.10)；

M107-amt-R：5′-gctctttatttcccggctgg-3′(SEQ ID NO.11)；

the target products with the sizes of about 1.1Kb and 3.1Kb are respectively obtained through PCR amplification, and the gene amt is mutated into a DNA sequence shown as SEQ ID NO. 16; completely accords with the theoretical expectation, and proves that the genome sequencing of the mutant strain is completely correct, and the mutant gene is identified without errors (figure 4).

Example 4

This example provides the construction of an amt gene phylogenetic tree.

According to the DNA sequence of the azotobacter GXGL-4A amt gene, genBank is submitted for BLAST comparison, a sequence with the sequence similarity of more than 80% is selected, and MEGA7 software is adopted to make a phylogenetic tree by an adjacent method (figure 5). The result shows that the gene has obvious species specificity, is highly conserved at the level in the species, has large gene sequence difference with the strains outside the Kosakonia species, and has long evolution distance.

Example 5

This example provides fluorescent quantitative PCR of mutant M107.

Taking azotobacter GXGL-4A as a reference, extracting total RNA of the strain by using a UNIQ-10 column type Trizol total RNA extraction kit, carrying out fluorescent quantitative PCR analysis on mutant M107 mutant genes after reverse transcription, and taking housekeeping gene rpoB as an internal reference gene. The fluorescent quantitative PCR primer sequence is as follows:

rpoB-F3：5'-GGTGCGTGTAGAGCGTGC-3'(SEQ ID NO.12)，

rpoB-R3:5'-ATCTCGGACAGCGGGTTG-3' (SEQ ID No. 13), PCR product: 168bp

amt-F4：5'-TCCATACGCCTGGGAAGTC-3'(SEQ ID NO.14)，

amt-R4:5'-AAGTTTCTGCCACTGTTCCG-3' (SEQ ID No. 15), PCR product: 178bp

(1) First Strand cDNA Synthesis

RNA was reverse transcribed at 800 ng.

1) The following reagents were added to ice-bath nucleo-free PCR tubes:

Random Primer p(dN)6(100pmol)1μL

dNTP Mix(0.5mM final concentration)1μL

Rnase-free ddH ₂ o constant volume is 14.5 mu L

2) Gently mixing, centrifuging for 3s, warm-bathing the reaction mixture at 65 deg.C for 5min, ice-bathing for 2min, and centrifuging for 3s;

3) The tube was iced in ice and the following reagents were added:

5x RT Buffer 4μL

Thermo Scientific RiboLock RNase Inhibitor(20U)0.5μL

Maxima Reverse Transcriptase(200U)1μL

4) Gently mixing and centrifuging for 3s;

5) The reverse transcription reaction was performed on a PCR instrument under the following conditions:

25℃，10min；50℃，30min；85℃，5min。

6) The solution was stored at-20 ℃.

(2) Fluorescent quantitative PCR

And diluting the cDNA sample by 10 times and using the diluted cDNA sample as a template to perform machine detection.

Quantitative PCR reagent: 2 xSG Fast qPCR Master Mix (B639271, BBI, roche).

Quantitative PCR instrument: lightCycler model 480 II fluorescent quantitative PCR instrument (Roche, rotkreuz, switzerland).

1) Preparing reaction mixed liquid

SybrGreen qPCR Master Mix(2×)5μL

Primer F (10. Mu.M) 0.2. Mu.L

Primer R (10. Mu.M) 0.2. Mu.L

ddH ₂ O 3.6μL

Template(cDNA)1μL

2) PCR cycling conditions

At 95 ℃ for 3min;45 cycles, each cycle comprising 95 ℃,5s;60 ℃ for 30s.

The 96/384 well plates with the added samples were placed in a LightCycler480 II (Roche) for reaction.

Fluorescent quantitative PCR detection showed that the mutated amt gene was hardly transcribed after insertion (FIG. 6). The expression product of the target gene has changed due to the mutation of Tn5 transposition.

Example 6

This example provides an assay for the growth curve of mutant M107.

After the azotobacter GXGL-4A and the mutant strain M107 are purified, single colonies are picked and placed in 20mL LB, and the colonies are placed on a shaking table and cultured overnight at 37 ℃ and 180 rpm. The activated strain was inoculated in 100mL LB at an inoculum size of 1% (V/V), and OD was measured 1 time every 1h ₆₀₀ Values, 3 replicates were performed for each treatment.

The results showed that the growth of the mutant strain of interest was hardly affected after gene mutation, and there was no significant difference in growth rate from the wild strain (fig. 7).

Example 7

This example provides an assay for cucumber growth promoting effect of mutant M107.

After the cucumber seedlings are treated by the target strains, the physiological indexes of the cucumber, such as plant height, fresh plant weight, root length, fresh root weight and the like, are measured. The results show that the biomass of the cucumber of the mutant M107 treatment group (CK) is obviously higher than that of the wild strain treatment group; in addition to root length, the other 3 biomass indices of the wild-treated group were significantly higher than those of the blank control group (sterile water treatment) (fig. 8), and fig. 9 shows the overall appearance of cucumber plants for each treatment. In a whole view, the azotobacter treatment has obvious growth promoting effect on cucumber plants, and the mutant strain M107 treatment can improve the growth vigor of the cucumber plants compared with wild plants.

Example 8

This example provides a measurement of the inhibition of the supernatant of the mutant M107 fermentation broth against pathogenic bacteria of northern leaf blight (Bipolaris maydis).

The strain was inoculated in LB medium and shaken overnight at 37 ℃. 50mL of the bacterial solution was centrifuged at 8000rpm for 5min. The supernatant was aspirated by a syringe, filtered through a microporous membrane, and the filtrate was stored in a 4mL centrifuge tube at 4 ℃ in a refrigerator for further use.

Taking 600 mu L of the filtrate, coating the filtrate on a PDA culture medium, adding an equal amount of liquid LB into a blank control, and uniformly coating. Then, the B.maydis lawn was inverted in the center of the PDA medium, lightly pressed several times to make it closely contact with the surface of the medium, inverted cultured in a constant temperature incubator at 28 ℃ for 4 days, and the lawn diameter was measured by the cross method. The results showed that the diameter of the lawn grown on the control plates was about 8cm, which was significantly higher than that of the M107 treated group. Azotobacter GXGL-4A and mutant M107 have obvious inhibition effect on growth of B.maydis, and the mutant M107 has good antibacterial effect. The siderophiles in the supernatant compete for the iron required for fungal growth, resulting in restricted growth of the pathogen of maculopathy, the higher the siderophiles content the slower the fungal growth (fig. 10).

The embodiments described above are described to facilitate an understanding and use of the invention by those skilled in the art. It will be readily apparent to those skilled in the art that various modifications to these embodiments may be made, and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above embodiments, and those skilled in the art should make improvements and modifications within the scope of the present invention based on the disclosure of the present invention.

Claims (10)

1. The application of a target gene in expressing azotobacter siderophore is characterized in that the target gene is an amt gene, and the DNA sequence of the amt gene is shown as SEQ ID NO. 1.

2. The application of the mutant M107 in expressing azotobacteria siderophore is characterized in that the preservation number of the mutant M107 is CGMCC No.24401.

3. The use of the mutant M107 strain of claim 2 for expressing azotobacter siderophin, wherein the method of using the mutant M107 strain for expressing azotobacter siderophin to promote plant growth comprises the steps of:

(1) Inoculating the mutant M107 into an LB culture medium, and collecting a mutant M107 cell precipitate after overnight culture;

(2) And (2) treating the mutant M107 cell sediment collected in the step (1) and applying the treated cell sediment to the rhizosphere of the plant.

4. The use of the mutant M107 according to claim 3 for expressing azotobacter siderophore, wherein in step (1), the mutant M107 is inoculated into LB medium at an inoculum size of 1%.

5. The use of the mutant M107 strain of claim 3 for expressing azotobacter siderophore, wherein in step (2), the post-treatment comprises resuspending the mutant M107 cell pellet in sterile water and then centrifuging, repeating the process for multiple times, and resuspending the mutant M107 cell pellet obtained from the last centrifugation in sterile water to obtain a mutant M107 cell suspension.

6. The use of mutant M107 according to claim 5 for expressing azotobacter siderophore, wherein the mutant M107 cell suspension is applied to the plant during the two-leaf one-heart stage.

7. The use of the mutant M107 strain of claim 6 for expressing azotobacteria siderophin, wherein 5-10mL of mutant M107 cell suspension is released per plant and the number of bacteria is 1X 10 ⁸ CFU/mL; the frequency was 1 application every 5 days with 5 consecutive treatments.

8. The application of a substance for knocking out or replacing a target gene in preparing a high-yield siderophin azotobacter or siderophin growth-promoting bacteria agent is characterized in that the target gene is an amt gene, and the DNA sequence of the amt gene is shown in SEQ ID NO. 1.

9. The application of the mutant M107 in preparing a high-yield azotobacter siderophilus strain or a growth-promoting strain for producing siderophilus is characterized in that the preservation number of the mutant M107 is CGMCC No.24401.

10. The mutant M107 is characterized in that the preservation number of the mutant M107 is CGMCC No.24401.

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