Method for constructing genetically engineered nitrogen-fixing bacterium with high-efficiency ammonia excretion

Abstract

Provided is a method for constructing a genetically engineered bacterium with high-efficiency ammonia excretion. Firstly, the coding sequence of the central region of the nitrogen fixation negative regulatory gene nifL of a nitrogen-fixing bacterium is knocked out. It is found that the engineered bacterium with the nifL gene mutation obtained by means of the method can realize high-efficiency nitrogen fixation and ammonia excretion under different nitrogen conditions, and the characteristics of high-efficiency nitrogen fixation and ammonia excretion are still retained after repeated subculture over successive years. Moreover, the normal expression of the electron transfer complex coding gene cluster rnf upstream of nifL and nifA downstream thereof is not affected after the central region of nifL is knocked out. The research shows that the key factor for realizing efficient nitrogen fixation and ammonia excretion of the engineered bacterium without introducing an exogenous promoter is the functional loss of nifL while ensuring no interference with the expression of genes upstream and downstream thereof. The method provided eliminates the requirement for site-directed insertion of an exogenous promoter, thus avoiding the problem that the function of the exogenous promoter of a mutant strain is prone to loss during repeated subculture over successive years. The obtained mutant strain has high-efficiency and stable nitrogen fixation performance.

Description

Construction method of genetically engineered nitrogen-fixing bacteria with high ammonia excretion

[0001] TECHNICAL FIELD

[0002] The present application relates to the field of nitrogen-fixing genetic engineering, and more particularly to a construction method of genetically engineered nitrogen-fixing bacteria with high ammonia excretion. BACKGROUND

[0003] Developing genetically engineered bacteria with high nitrogen fixation is an important strategy for developing low-carbon green agriculture. Almost all nitrogen fixation-related genes (nif) of Prokaryotes require NifA to activate transcription, and the negative regulation protein NifL can inhibit the activity of NifA by binding to it, limiting the nitrogen fixation of the strain. Removing the nifL negative regulation of autotrophic nitrogen-fixing bacteria can significantly improve the nitrogen fixation and ammonia excretion of the engineered bacteria. In theory, complete deletion of nifL or changing the topological relationship between the structural domains of nifL to maintain a conformation that cannot bind NifA can remove the negative regulation of nitrogen fixation of the strain, allowing the engineered bacteria to have a sustained nitrogen fixation and ammonia excretion phenotype (Nif+ / (NH4+)+). However, the several nifL mutant engineered bacteria developed so far are limited by heterologous promoters. A reverse promoter must be inserted at the nifL deletion site to make the strain exhibit the Nif+ / (NH4+)+ phenotype. Neither deletion of the coding sequence of different domains of nifL nor complete deletion of nifL can achieve high-efficiency nitrogen fixation and ammonia excretion of the strain. Current research suggests that inserting a suitable exogenous reverse promoter at the nifL knockout site to enhance the expression of the upstream electron transport complex gene cluster rnf is the key to constructing high-efficiency nitrogen-fixing engineered bacteria. However, exogenous DNA can lose function over time in the absence of selective pressure, and promoters in genetic circuits are most susceptible to loss-of-function mutations. Therefore, the genetic instability of current nifL mutant engineered bacteria may be a major obstacle to their practical application. Patent CN117126793A discloses a high-ammonia-excreting Rhodopseudomonas azotoformans genetically engineered bacterium and its application. Specifically, a high-ammonia-excreting Rhodopseudomonas azotoformans genetically engineered bacterium a4 was obtained by knocking out the nifL negative regulation gene of wild Rhodopseudomonas azotoformans. However, there is currently a lack of a construction method for obtaining a stable genetically engineered nifL mutant bacteria with high nitrogen fixation. SUMMARY

[0004] The present application aims to overcome the above-mentioned defects and deficiencies in the prior art and provide a construction method of genetically engineered nitrogen-fixing bacteria with high ammonia excretion.

[0005] The above-mentioned object of the present application is achieved by the following technical solution:

[0006] The present application is based on the scarless gene knockout technology of homologous recombination to knockout the nitrogen fixation negative regulation protein coding gene nifL of Azotobacter chroococcum CICC® 22663, and the functional domains of nifL are predicted by using SMART and AlphaFold3. The prediction results show that nifL contains a typical amino-terminal PAS domain (including PAS1 25-91, PAC 97-139, PAS2 151-271) responding to intracellular signal molecules and a carboxy-terminal GHKL domain (404-517), as well as a central region without clear structural homologues (helical structure containing a relatively conserved linker LCR (282-294)). Therefore, a plurality of pairs of primers are designed at the positions of different domains of nifL by using Primer Premier 5.0 to knockout nifL in segments, and the nitrogen fixation and ammonia excretion performance of each nifL mutant strain is detected by indigo blue colorimetry. The present application finds that the high-efficiency nitrogen fixation engineering strain A4 with the central region of nifL deleted in a plurality of nifL domain deletion mutant strains, compared with other nifL mutant engineering strains reported at present, the A4 strain obtains a high-efficiency nitrogen fixation phenotype without introducing any exogenous gene; compared with the engineering strains with the amino-terminal and carboxy-terminal of nifL deleted, the A4 strain shows high-efficiency and stable nitrogen fixation and ammonia excretion characteristics. At the same time, the RNA-seq results of different mutant strains show that, compared with other scarless deletion mutant strains, the strain with the central region of nifL deleted does not affect the normal expression of the electron transport complex coding gene cluster rnf upstream of nifL and nifA downstream, and can normally and efficiently fix nitrogen. The present application research shows that the key factor to realize high-efficiency nitrogen fixation and ammonia excretion of the engineering strain without introducing an exogenous promoter is the functional deletion of nifL without interfering with the expression of the upstream and downstream genes. Therefore, the above method does not need to insert an exogenous reverse promoter at the nifL knockout site, which can avoid the genetic instability problem existing in the nifL mutant engineering strains of introducing exogenous DNA.

[0007] Therefore, the present application provides a construction method of a high-efficiency ammonia excretion gene engineering nitrogen fixation strain, which is to knockout the central region coding sequence of the nitrogen fixation negative regulation gene nifL of the nitrogen fixation strain, and to screen a mutant strain with the central region of nifL knocked out without affecting the normal expression of the electron transport complex coding gene cluster rnf upstream of nifL and nifA downstream. Since the existing gene knockout technology cannot completely guarantee the precise knockout of the target region, deviations may occur, for example, the amino-terminal and carboxy-terminal of nifL may also be knocked out, so it is still necessary to screen a mutant strain with the central region of nifL knocked out without affecting the normal expression of the electron transport complex coding gene cluster rnf upstream of nifL and nifA downstream.

[0008] Further, the knockout is a scarless gene knockout technology using homologous recombination.

[0009] Further, the scarless gene knockout technology is a technology based on the principle of homologous recombination, in which primers for gene knockout are designed in the upstream and downstream of the central region of the nitrogen fixation negative regulatory gene nifL, a suicide plasmid is used as a carrier to construct a recombination plasmid, the nitrogen fixation bacteria are transformed, transformants are screened, and a positive mutant strain with the central region of nifL knocked out and the normal expression of the electron transport complex coding gene cluster rnf in the upstream of nifL and nifA in the downstream is obtained.

[0010] Further, the suicide vector is pK18mobsacB or pEX18Gm.

[0011] Further, the primers for gene knockout are designed in the position 500-1000 bp in the upstream and downstream of the central region of the nitrogen fixation negative regulatory gene nifL.

[0012] Further, the endonuclease used in the construction of the recombination plasmid is BamH I and Sal I.

[0013] Further, the ligase used in the construction of the recombination plasmid is T4 DNA Ligase.

[0014] Further, the method for transforming the nitrogen fixation bacteria is an electroporation method.

[0015] Further, the method for screening the transformants is to amplify the target fragment by PCR, perform sequencing, and finally detect the expression of rnf and nifA by the method of RNA-seq.

[0016] Further, the method for screening the transformants is to first screen the homologous recombination single exchange bacteria using solid culture medium containing antibiotics, screen the homologous recombination double exchange bacteria using solid culture medium containing sucrose, amplify the target fragment by PCR, perform sequencing, and finally detect the expression of rnf and nifA by the method of RNA-seq.

[0017] Further, the nitrogen fixation bacteria are self-nitrogen fixation bacteria containing the nifL gene.

[0018] Further, the nitrogen fixation bacteria are Azotobacter chroococcum.

[0019] Preferably, the Azotobacter chroococcum is wild Azotobacter chroococcum 22663.

[0020] Further, the nucleotide sequence of the coding sequence of the central region of nifL of the Rhizobium rhizomosarum is shown in SEQ ID No. 1.

[0021] Further, the sequences of the knockout primers of the central region of nifL of the Rhizobium rhizomosarum are shown in SEQ ID No. 2-5.

[0022] The application also provides a high-efficiency ammonia-secreting genetically engineered nitrogen-fixing bacterium prepared by the construction method.

[0023] Compared with the prior art, the application has the following beneficial effects:

[0024] The application provides a construction method of a high-efficiency ammonia-secreting genetically engineered bacterium. First, the central region of a nitrogen-fixing negative regulation gene nifL of a nitrogen-fixing bacterium is knocked out. It is found that the engineered bacterium with the central region of the nifL gene mutated by the method can realize high-efficiency nitrogen fixation and ammonia secretion under different nitrogen conditions, and still retains the high-efficiency nitrogen fixation and ammonia secretion characteristics after continuous multiple generations of subculture for many years. Moreover, the normal expression of an electron transfer complex coding gene cluster rnf upstream of the nifL and nifA downstream of the nifL is not affected after the central region of the nifL is knocked out. It is indicated that the key factor to realize high-efficiency nitrogen fixation and ammonia secretion of the engineered bacterium without introducing an exogenous promoter is that the nifL function is lost and the upstream and downstream gene expressions are not disturbed. The method provided by the application does not need to insert an exogenous promoter at a fixed point, avoids the problem that the function of the exogenous promoter of the mutant strain is easily lost in the process of subculture for many years, and obtains a mutant strain with high-efficiency and stable nitrogen fixation performance. The field test results also show that the engineered bacterium with the central region of the nifL gene mutated can significantly improve the biomass of plants. BRIEF DESCRIPTION OF DRAWINGS

[0025] FIG. 1 is a schematic diagram of a recombinant suicide plasmid for A4 gene knockout.

[0026] FIG. 2 is the position of the conserved domain of the gene nifL in Ac and the deletion position of four nifL mutant strains.

[0027] FIG. 3 is that the mutant bacteria A2 and A3 flocculate and precipitate in the Ashby nitrogen-free medium, and cannot normally proliferate.

[0028] FIG. 4 is that the mutant strain Z1 normally grows in the Ashby nitrogen-free medium in the initial stage. The middle and right graphs are the mutant bacteria A2 / A3 inoculated at the same time, and the left graph is the mutant strain Z1.

[0029] FIG. 5 is that the mutant strain Z1 is preserved and then reactivated and cultured. The strain after reactivation cannot normally grow in the nitrogen-free medium. The left graph is the normal nitrogen-fixing bacterium inoculated at the same time, and the right graph is the mutant strain Z1.

[0030] Figure 6 is a schematic diagram of the SMART domain search results of all 485-525 aa NifL protein sequences in the NCBI database. Embodiments of the present application

[0031] The present application is further illustrated by the following description with reference to the accompanying drawings and specific examples. The examples do not limit the present application in any form. Unless otherwise specified, the reagents, methods and equipment used in the present application are conventional reagents, methods and equipment in the technical field.

[0032] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0033] Example 1 Construction of high ammonia-secreting Azotobacter chroococcum genetically engineered bacteria

[0034] 1. Experimental methods

[0035] (1) Primer design

[0036] The full-length nifL is 1560 bp, encoding 519 amino acids. The functional domains of nifL were predicted using SMART, and the prediction results showed that it contains a typical amino-terminal signal domain (PAS1 25-91, PAC 97-139, PAS2 151-271), a carboxy-terminal GHKL domain (404-517), and a central region containing a conserved linker structure flexible linker LCR (282-294). Using Primer Premier 5.0, a plurality of primers were designed according to the positions of 500-1000 bp upstream and downstream of different domains of wild-type Azotobacter chroococcum CICC® 22663 (Ac) (reference the multiple cloning site information of the suicide plasmid vector and the T cloning vector (pTA2), select appropriate restriction enzymes, double enzyme cut the suicide plasmid vector and the T vector cloned with the upstream and downstream genes of the target fragment, or directly add appropriate enzyme cutting sites (Z1 mutation) to the 5' end of the primer), for the partitioned knockout of nifL gene.

[0037]

[0038] (2) Vector construction of A2, A3, and A4 strains

[0039] The upstream and downstream homologous arm amplification is performed by using the above primers, the overlapping extension PCR technology is used to connect the upstream and downstream homologous arms, the target fragment is obtained, and the target fragment is cloned into a T vector (pTA2). The T cloning vector containing the target fragment and the suicide vector are subjected to restriction endonuclease BamH I and Sal I digestion, the target fragment containing the sticky end of the endonuclease and the linearized pK18mobsacB vector are obtained, the target fragment is connected with the linearized vector by using T4 DNA ligase, and the recombination vector required for scarless gene knockout is obtained. The recombination vector is electroporated into the wild-type Azotobacter chroococcum CICC® 22663 strain.

[0040] (3) Vector construction of the Z1 strain

[0041] The pEX18Gm is used as a vector, the construction is performed according to the method in (2), and the same length of a kanamycin resistance box and a forward promoter sequence are inserted into the deletion site. The Z1-kanR-F1 / R1 primer is used to clone the kanamycin resistance gene (KIXX) from the pET28a-EGFP vector, and then the overlapping extension PCR technology is used to connect the upstream and downstream homologous arms and the cloned KIXX, so as to obtain the target fragment (i.e., the upstream gene of the Z1 deletion region + KIXX + the downstream gene of the Z1 deletion region) for Z1 mutation. The target fragment and the suicide vector pEX18Gm are subjected to restriction endonuclease BamH I and Sal I digestion, and then the target fragment is connected with the linearized vector according to the method in (2) and is electroporated into the wild-type strain, so as to obtain the mutant strain in which the KIXX is inserted.

[0042] (4) Result detection

[0043] The homologous recombination single exchange strain is screened by using the solid SOC culture medium containing kanamycin (or gentamicin), the homologous recombination double exchange strain is screened from the single exchange strain by using the solid SOC culture medium containing 15% sucrose, the corresponding gene deletion mutant strain is screened from the double exchange strain by using the gene knockout verification primer, the PCR positive strain is sent to Shengong for sequencing, the sequencing result is compared with the nifL gene sequence of the wild-type strain, and whether the target deletion fragment is successfully knocked out is further verified.

[0044] 2, Experimental results

[0045] The results show that according to the above experimental method, four Ac mutant strains with different deletion sites are obtained, and are named as A2, A3, A4 and Z1.

[0046] The A2 strain is a mutant strain in which the nifL amino-terminal PAS1 domain (8-406 bp) is deleted;

[0047] A3 strain is a mutant strain with deletion of nifL amino-terminal PAS2 domain (356-782 bp);

[0048] A4 strain is a mutant strain with deletion of nifL central region (748-1159 bp), and the schematic diagram of the knock-out recombination vector is shown in Figure 1.

[0049] Z1 strain is a mutant strain with deletion of most of the sequence (364-1302 bp) and insertion of a kanamycin resistance gene and its forward promoter sequence of the same length at the deletion site.

[0050] The deletion positions of the four nifL gene deletion Ac mutant strains are shown in Figure 2.

[0051] Example 2: Nitrogen fixation effect and stability detection of the four nifL gene deletion Ac mutant strains

[0052] 1. Experimental method

[0053] (1) The nitrogen fixation effect of the four nifL gene deletion Ac mutant strains was monitored continuously for several days by indigo blue colorimetry. The basic principle of indigo blue colorimetry is that NH 4+ , reacts with hypochlorite and phenol in a strong alkaline medium to form water-soluble dye indigo blue. The deeper the color of the sample, the higher the ammonium nitrogen content in the sample. Different strains were incubated in a nitrogen-free liquid medium, after a period of time, the bacterial liquid was collected, centrifuged and the supernatant was filtered through a 0.45 micron filter membrane to obtain the extracellular solution of the culture system. The stronger the nitrogen fixation and ammonia excretion performance of the strain, the higher the accumulation of ammonium nitrogen in the extracellular solution. The nitrogen fixation and ammonia excretion performance of different strains was judged by the accumulation of ammonium nitrogen in the extracellular solution of the culture system. High ammonium nitrogen accumulation strains, i.e. high-efficiency nitrogen fixation strains, were screened.

[0054] Prepare reaction liquid A: 1.25% sodium nitroprusside solution 4 mL, 10.00 g phenol, constant volume to 1000 mL (4°C storage, red brown color not available).

[0055] Prepare reaction liquid B: 5.0 g sodium hydroxide, 4.0 g trisodium citrate, 7 mL sodium hypochlorite solution, constant volume to 1000 mL (4°C storage)

[0056] Test solution: inoculate nitrogen fixation bacteria into nitrogen-free medium and shake culture for 24 h or more. Take 1 ml of bacterial liquid in a 1.5 ml centrifuge tube, centrifuge at 5000 rpm for 3 min, inject the supernatant with a syringe, filter with a 0.45 micron filter membrane, and store the filtrate in a new centrifuge tube, which is the test solution (extracellular solution).

[0057] Detection: 5 mL of A solution and B solution, 100 μL of the test solution, mixed uniformly in the reagent bottle, reacted at 37°C for 30 min, and observed whether the solution turned blue (qualitative); 100 μL of the reaction solution was taken into a 96-well plate, and the absorbance at 637 nm was detected by an enzyme-labeled instrument (quantitative). At the same time, the standard solution of ammonia nitrogen was used: GSB 05-1145-2000 environmental standard sample (500 mg / L 102224), and standard solutions with different ammonia nitrogen concentrations were prepared, the absorbance at 637 nm was detected, and a standard curve was drawn.

[0058] (2) Detection of nitrogen fixation and ammonia excretion of the mutant strain after multiple passages and preservation and reactivation:

[0059] After the initial shake flask detection on July 18, 2021, the strains successfully constructed and screened were preserved in a 4°C refrigerator or a -80°C refrigerator. The strains preserved in the 4°C refrigerator were subcultured once every 15 days on average, and after multiple passages, the strains were transferred to a -80°C refrigerator for long-term preservation. The strains preserved in the -80°C refrigerator were subcultured once every 6 months on average. The strains were extracted every year during the subculture period to detect their nitrogen fixation and ammonia excretion performance, and the high-efficiency nitrogen fixation phenotype was detected after continuous 3-year multiple passage culture, preservation and reactivation.

[0060] 2. Experimental results

[0061] (1) The four nifL gene deletion Ac mutant strains were cultured in nitrogen-free medium (Ashby, Burk's), and the results are shown in FIG. 3. Mutant strains A2 and A3 flocculated in the medium and could not proliferate normally (nitrogen fixation phenotype was lost). As shown in Table 1, mutant strain A2 recovered the nitrogen fixation performance after 4 days of continuous culture, but still had no high-efficiency nitrogen fixation and ammonia excretion phenotype. As shown in FIG. 4, mutant strain Z1 could grow normally in the nitrogen-free medium at the initial stage, and although it retained the nitrogen fixation performance, its ammonia excretion level was equivalent to that of the wild-type strain Ac and had no significant improvement. As shown in FIG. 5, after preservation and re-culture, it was found that mutant strain Z1 could not grow normally on the nitrogen-free solid medium, indicating that it lost the nitrogen fixation performance.

[0062] Table 1. Detection of nitrogen fixation performance of mutant strain A2

[0063]

[0064] (2) The nitrogen fixation and ammonia excretion levels of mutant strain A4 were detected in different culture systems a. nitrogen-free treatment; b. nitrate nitrogen treatment: 10 mM nitrate nitrogen; c. urea treatment: 10 mM urea. As shown in Table 2, the comparison of ammonia nitrogen content showed that the nitrogen fixation performance of mutant strain A4 was significantly better than that of the wild-type strain, and the additional supply of common nitrogen nutrients did not affect the continuation of the nitrogen fixation reaction. The specific nitrogen fixation performance test results are as follows:

[0065] In 200 mL of burk's nitrogen-free culture system, the extracellular ammonia nitrogen concentration of mutant A4 can reach 27 mM, and its ammonia nitrogen content accumulates day by day from the first day of monitoring. Under the same conditions, the detection value of the extracellular ammonia nitrogen of the wild strain fluctuates every day, and there is no accumulation trend (the detection value is high and low, which may be that the ammonia nitrogen secreted into the extracellular is then absorbed and utilized by the wild strain itself), and the highest concentration is only 0.14 mM. The ammonia secretion amount of the mutant strain is about 190 times that of the wild type under experimental conditions.

[0066] In the burk's culture system supplemented with 10 mM nitrate nitrogen, the extracellular ammonia nitrogen concentration of the mutant bacteria can reach 19 mM, and its ammonia nitrogen content increases day by day from the first day. In the presence of 10 mM nitrate nitrogen, the extracellular ammonia nitrogen content of the wild type only reaches about 2 mM in the first two days, and then its ammonia nitrogen content fluctuates around 0.3 mM.

[0067] In the burk's culture system supplemented with 10 mM urea, the extracellular ammonia nitrogen concentration of the mutant bacteria can reach 37 mM, and 10 mM urea can generate 5.67 mM ammonia nitrogen after hydrolysis, so the extracellular secretion of ammonia nitrogen concentration of the mutant bacteria can reach 31.33 mM. The ammonia nitrogen content in the culture medium increases day by day from the first day, and is much higher than the added urea concentration, and reaches a stable value on the seventh day. After the wild type is supplemented with 10 mM urea, the ammonia nitrogen content in the culture system reaches a peak value (about 13 mM) on the second day, and then its extracellular ammonia nitrogen content gradually decreases and fluctuates around 2 mM; the ammonia nitrogen concentration is always lower than the added urea nitrogen concentration, mainly due to the absorption of the added nitrogen source by the bacteria. In the treatment group supplemented with urea nitrogen, the ammonia nitrogen content of the wild type and the engineered strain is higher than that in other culture groups. This part of ammonia nitrogen does not come from the nitrogen fixation reaction of the strain, but is produced by the urease of the nitrogen-fixing bacteria to decompose urea.

[0068] Table 2 Comparison of nitrogen fixation performance of mutant A4 and wild type Ac in different nitrogen culture systems

[0069]

[0070] In summary, the nitrogen fixation performance of the mutant strain A4 of Rhizobium rhizomosarum is significantly better than that of the wild type, and the additional supplementation of commonly used nitrogen nutrients does not affect the continuation of its nitrogen fixation reaction. The ammonia nitrogen concentration in the nitrogen-free culture medium can reach 27 mM, which is significantly higher than that of existing nitrogen fixation mutant strains, and the engineered strain does not introduce any exogenous genes.

[0071] (3) The results of detecting the nitrogen fixation and ammonia secretion of the mutant strain A4 after multiple passages and preservation are shown in Table 3. The ammonia nitrogen accumulation level of the mutant strain A4 and the wild type Ac in the nitrogen-free culture system after multiple passages was detected by sampling, and the results showed that there was no significant change in the nitrogen fixation and ammonia secretion yield of the A4 strain passed from 2021 to 2024, and its high-efficiency nitrogen fixation and ammonia secretion performance was stable.

[0072] Table 3 Ammonia nitrogen accumulation levels of mutant bacteria A4 and wild bacteria Ac in nitrogen-free culture system after multiple passages

[0073]

[0074] In summary, the application obtains a high-efficiency ammonia-secreting and non-foreign gene-introduced Rhizobium sp. A4 by screening after segmentally knocking out the nifL gene of wild Rhizobium sp.

[0075] Example 3 Expression of the upstream gene cluster rnf and the downstream gene nifA of nifL

[0076] In combination with the results of Example 2, only the nifL central region deletion strain can produce high-efficiency ammonia secretion effect. To explore the differences in the nitrogen fixation effect and stability of the above four nifL gene deletion Ac mutant strains, the application performs transcriptome sequencing on the above four mutant strains and wild bacteria, wherein the expression of the upstream gene cluster rnf and the downstream gene nifA of nifL is shown in Table 4.

[0077] Table 4 Expression of the gene cluster rnf and the gene nifA in different nitrogen-fixing bacteria (FPKM)

[0078]

[0079] Note: N=3 in mutant strain A4 and wild bacteria Ac; N=1 in mutant strain A2 and mutant strain A3.

[0080] In summary, through transcriptome data analysis, the main reason why other region deletions (A2, A3, Z1) cannot obtain high-efficiency nitrogen-fixing bacteria is that the DNA conformation changes caused by these region deletions interfere with the binding of RNA polymerase or activator to the promoter region or upstream activation sequence (this part of DNA element is located in the non-coding region between rnf and nifL genes) in front of rnf, thereby interfering with the normal expression of rnf; at the same time, the deletion of these regions also affects the expression of the downstream gene nifA of nifL, resulting in low nitrogen fixation or no nitrogen fixation effect of the nitrogen-fixing bacteria. The deletion region of mutant strain A4 is located in the center of nifL gene, far away from the functional non-coding region, and the DNA conformation change caused by the deletion of this part does not or is not enough to interfere with the normal expression of the upstream and downstream genes of the deleted gene, so the upstream rnf and downstream nifA are normally expressed, and the nifL negative regulation function is lost, so the overall nitrogen fixation performance is improved.

[0081] Meanwhile, as shown in Figure 6, all NifL protein sequences with the length of 485-525 aa downloaded from NCBI, and using SMART query protein domain, the results show that NifL protein generally has N-terminal PAS domain and C-terminal GHKL domain and central ambiguous domain, especially azotobacter bacteria, so that the NifL protein is highly conserved in different nitrogen-fixing bacteria. In conclusion, the construction method of the high-efficiency ammonia-secreting genetically engineered nitrogen-fixing bacteria provided by the application knocks out the central region coding sequence of the nitrogen-fixing negative regulation gene nifL of the nitrogen-fixing bacteria, and does not affect the normal expression of the electron transfer complex coding gene cluster rnf upstream of nifL and nifA downstream, has universal applicability, and different types of high-efficiency ammonia-secreting genetically engineered nitrogen-fixing bacteria can be obtained based on the method.

[0082] Example 4 Field test of the growth-promoting effect of the nitrogen-fixing mutant bacteria

[0083] A field plot experiment was used to verify the stability and nitrogen-fixing growth-promoting effect of the A4 engineering strain under actual production conditions, and the experimental site was in the Panyu campus of Jinan University in Guangzhou. Three controls of 40, 50 and 60 mg / kg of urea nitrogen fertilizer were set, and two treatments of single inoculation and inoculation + 10 mg / kg of urea nitrogen fertilizer were set. Each control and treatment was set in 3 plots in parallel, and each plot had an area of 2 square meters. The background available nitrogen content of the surface soil of each control and treatment before sowing was basically consistent (see Tables 5 and 6). After the seeds of Euphorbia humilis were disinfected, they were uniformly sown in each plot, and the seedlings were thinned to about 130 per square meter after 4 leaves. The seeds were sown on April 25, the bacteria were inoculated and the fertilizer was applied on May 20, the inoculation method was to inoculate 250 mL of bacterial liquid with an OD600 value of 0.6-0.7 to the rhizosphere of the crops per square meter, the fertilizer application method was to uniformly spread urea in the plot, and the plants were harvested on June 15. The soil nitrogen detection sampling method before sowing and after harvesting the crops was to randomly collect 7 soil samples with a depth of 0-15 cm from each plot, uniformly mix them into one combined sample for detection, and represent the soil nitrogen content of the plot. The calculation method of the urea nitrogen replaced by inoculation was:

[0084] The difference between the nitrogen consumed by the control and the nitrogen consumed by the inoculation treatment was multiplied by the yield increase multiple of the inoculation, that is:

[0085] The urea nitrogen replaced by inoculation = ((the soil available nitrogen before sowing of the control + the nitrogen of the control after applying fertilizer - the soil available nitrogen after harvesting of the control) - (the soil available nitrogen before sowing of the treatment + the nitrogen of the treatment after applying fertilizer - the soil available nitrogen after harvesting of the treatment)) * (the biomass of the treatment / the biomass of the control)

[0086] The results are shown in Table 5 and Table 6, and the biomass of the pure inoculation treatment of Jiliayacai is 1.60 times, 1.46 times and 1.27 times of the respective fertilization control, which is equivalent to 76.4-80.9 mg / kg of urea nitrogen in the root layer soil replaced by the pure inoculation. The biomass of the inoculation + small amount of nitrogen fertilizer treatment is 2.07 times, 1.89 times and 1.64 times of the respective fertilization control, and the biomass reaches an extremely high yield of 37.8 tons per hectare, which is equivalent to 82.4-90.2 mg / kg of urea nitrogen in the root layer soil replaced by the inoculation.

[0087] Table 5 Comparison of biomass and nitrogen utilization of Jiliayacai in the field under different inoculation and nitrogen treatments

[0088]

[0089] Table 6 Replacement of urea nitrogen in the root layer soil by Jiliayacai inoculation

[0090]

[0091] In summary, the nifL engineering strain A4 with deletion of the central region of the nifL gene constructed in the application shows stable growth-promoting effect in actual production, can replace 76.4-80.9 mg / kg of urea nitrogen under the condition of single inoculation without supplement of nitrogen fertilizer, and can replace 82.4-90.2 mg / kg of urea nitrogen under the condition of inoculation with supplement of a small amount of nitrogen fertilizer. The engineering strain with deletion of the central region of the nifL gene does not introduce any exogenous gene, and shows significant growth-promoting effect in the field planting of Jiliayacai. The supplement of a small amount of nitrogen fertilizer does not interfere with the growth-promoting effect of the strain itself, but enhances the growth-promoting performance. It can be seen that the mutant strain provided in the application, which is obtained by knocking out the central region sequence of the nifL gene of the nitrogen-fixing bacteria, can efficiently fix nitrogen and has stable phenotype under field conditions.

Claims

1. A method for constructing a genetically engineered nitrogen-fixing bacterium with high ammonia excretion, characterized by, The construction method is to knock out the coding sequence of the central region of the nitrogen fixation negative regulation gene nifL of the nitrogen-fixing bacteria, and screen the mutant strain with the central region of nifL knocked out and normal expression of the electron transport complex coding gene cluster rnf upstream of nifL and nifA downstream of nifL.

2. The method of claim 1, wherein, The knocking out is a scarless gene knocking out technology by homologous recombination.

3. The method of claim 2, wherein, The scarless gene knocking out technology is to design primers for gene knocking out based on the principle of homologous recombination at the upstream and downstream of the central region of the nitrogen fixation negative regulation gene nifL, use a suicide plasmid as a carrier to construct a recombination plasmid, transform the nitrogen-fixing bacteria, screen the transformants, and obtain the positive mutant strain with the central region of nifL knocked out and normal expression of the electron transport complex coding gene cluster rnf upstream of nifL and nifA downstream of nifL.

4. The method of claim 3, wherein, The suicide vector is pK18mobsacB or pEX18Gm.

5. The construction method according to claim 3, characterized in that, The primers for gene knocking out are designed at the position 500-1000 bp upstream and downstream of the central region of the nitrogen fixation negative regulation gene nifL.

6. The method of construction according to claim 3, wherein, The method for screening the transformants is to amplify the target fragment by PCR, sequence, and then detect the expression of rnf and nifA by the method of RNA-seq.

7. The method of claim 1, wherein, The nitrogen-fixing bacteria are the free-living nitrogen-fixing bacteria containing the nifL gene.

8. The method of construction according to claim 7, wherein, The free-living nitrogen-fixing bacteria are Azotobacter chroococcum.

9. The method of construction according to claim 8, wherein, The nucleotide sequence of the coding sequence of the central region of nifL of the Azotobacter chroococcum is shown in SEQ ID No.

1.

10. The method of claim 9, wherein, The sequences of the knocking out primers of the central region of nifL of the Azotobacter chroococcum are shown in SEQ ID No. 2-5.

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