Construction method and application of sphingomonas ncd2 gene overexpression strain

Abstract

The invention discloses a construction method and application of a sphingomonas ncd2 gene overexpression strain, and the ncd2 gene overexpression strain is formed by introducing an ncd2 gene into sphingomonas through a recombinant plasmid or integrating the ncd2 gene onto a chromosome of sphingomonas through a genetic engineering means. The construction method comprises the steps of designing an expression vector of the ncd2 gene, cloning the ncd2 gene, transforming sphingomonas, carrying out resistance screening, verifying expression of the ncd2 gene and optimizing culture conditions, after the ncd2 gene is applied to the broiler chicken, the intestinal microbial flora composition and the intestinal histological structure can be improved, meanwhile, the contents of total nitrogen, uric acid and ammonium nitrogen in the chicken manure of the broiler chicken can be remarkably reduced, and the yield of the broiler chicken is improved. The content of nitrate nitrogen is obviously increased, so that the slaughtering rate can be increased, and the economic benefit is further increased.

Description

Technical Field

[0001] The present invention belongs to the field of microbial genetic engineering. Specifically, it relates to a method for constructing an overexpression strain of the ncd2 gene of Sphingomonas and its application. Background Art

[0002] Under the stimulation and induction of high-concentration ammonium sulfate, the expression of the ncd2 gene in the nitrogen metabolism pathway of Sphingomonas is significantly upregulated. The enzyme encoded by this gene is NMO, which is a flavin mononucleotide-dependent enzyme that oxidizes P3N and can catalyze the formation of nitrite from nitroalkanes (Zhang et al 2021). Studies have shown that overexpressing the ncd2 gene in Bacillus can completely degrade nitroethane, and the amount of cofactor FMN is crucial for the degradation of nitroalkanes by the ncd2 overexpression strain. In addition, adding FMN to the cell-free extract can significantly improve the degradation efficiency of nitroalkanes, up to two to three times. Currently, nitro monooxygenase has been found in a variety of microorganisms, such as Neurospora crassa, Pseudomonas aeruginosa, Bacillus, etc.

[0003] However, how to construct overexpression and knockout strains of the ncd2 gene, verify the effect of this gene on the ammonia degradation ability of Sphingomonas, optimize its culture conditions, and also consider the application of the Sphingomonas ncd2 gene overexpression strain are problems that need to be solved. Summary of the Invention

[0004] In view of the above defects, the present invention provides an overexpression strain of the ncd2 gene of Sphingomonas, named the overexpression strain of the ncd2 gene of Sphingomonas. The overexpression strain of the Sphingomonas ncd2 (Sphingomonas sp.) gene is deposited in the China Center for Type Culture Collection, with the deposit date of March 18, 2025, and the deposit number of CGMCC No. 33862.

[0005] The present invention discloses a method for constructing an overexpression strain of the ncd2 gene of Sphingomonas, comprising the following steps:

[0006] S1. Determine the resistance characteristics of Sphingomonas by measuring the minimum inhibitory concentration with appropriate antibiotics.

[0007] S2. First, extract genomic DNA from Sphingomonas, amplify the ncd2 gene using specific primers, and then perform Gibson assembly with the linearized vector to construct a recombinant plasmid.

[0008] S3. Transform the recombinant plasmid into competent cells, and through heat shock and recovery culture, make it grow on the selective medium, and finally screen out the successfully transformed strains.

[0009] S4. Culture Sphingomonas, prepare competent cells, transform the plasmid into these cells, and then screen for successfully transformed clones on a selective medium;

[0010] S5. Extract genomic DNA from the transformed strain and perform PCR verification to confirm the successful overexpression of the ncd2 gene;

[0011] S6. Optimize the culture conditions in the obtained ncd2 overexpressing strain.

[0012] Furthermore, step S1 specifically includes:

[0013] S101: Select gentamicin, streptomycin, and tetracycline to determine the minimum inhibitory concentration of Sphingomonas;

[0014] S102: Inoculate a certain volume of Sphingomonas in each well and culture overnight at 37°C;

[0015] S103: Measure the absorbance value of each well at a wavelength of 600 nm using a spectrophotometer.

[0016] Furthermore, step S2 specifically includes:

[0017] S201: Extract the genomic DNA of Sphingomonas, and amplify the ncd2 gene fragment containing the P1600-gent-Flag homologous arm using RBSF and pFLP3-RRNBTR primers;

[0018] S202: Perform gel electrophoresis on the amplification product and use an agarose gel recovery kit to recover the target product;

[0019] S203: Use NcoI and EcoRI to digest the p1600-gent-Flag vector backbone and incubate at 37°C for 1 hour to linearize the vector;

[0020] S204: Use Gibson assembly to recombine the linearized vector and the ncd2 gene fragment containing the homologous arm, and incubate at 50°C for 30 minutes.

[0021] Furthermore, step S3 specifically includes:

[0022] S301: Thaw DH5α competent cells on ice, add 10 μL of the recombinant product to 100 μL of competent cells, flick the tube wall gently to mix, and let stand on ice for 30 minutes;

[0023] S302: After heat shock in a 42°C water bath for 45 seconds, immediately place on ice and cool for 2 - 3 minutes;

[0024] S303: Add LB medium without adding antibiotics, shake the bacteria at 37 °C for 1 hour at a rotation speed of 200 - 250 rpm;

[0025] S304: Preheat an LB solid medium plate containing 50 μg / mL gentamicin in a 30 °C incubator.

[0026] S305: Centrifuge at 5000 rpm for 5 minutes, discard 900 μL of the supernatant, resuspend the bacterial cells, and gently spread them evenly on a plate containing 50 μg / mL Gm, and incubate them upside down in a 37 °C incubator for 12 - 16 hours;

[0027] S306: Pick a monoclonal colony on the recombinant reaction transformation plate for colony PCR identification.

[0028] Further, the specific steps of step S4 include:

[0029] S401: Inoculate a single colony from a freshly cultured plate into 3 mL of LB liquid medium, and culture it at 200 rpm and 37 °C for 16 hours;

[0030] S402: Inoculate 1.5 mL of the overnight cultured bacterial solution into 150 mL of fresh LB medium, and culture it at 200 rpm and 37 °C until the number of bacteria reaches approximately 1×10 8 CFU / mL;

[0031] S403: Transfer the bacterial solution to a 50 mL centrifuge tube, ice-bath for 15 minutes, then centrifuge at 4 °C and 4500 rpm for 5 minutes to collect the bacterial cells;

[0032] S404: Discard the supernatant, resuspend the bacterial cells with 30 mL of ice-cold 10% glycerol, ice-bath for 5 minutes, then centrifuge at 4 °C and 4500 rpm for 5 minutes to collect the bacterial cells;

[0033] S405: Resuspend the bacterial cells with 2 mL of pre-cooled 10% glycerol, with a cell concentration of 1×10 10 CFU / mL, aliquot into 1.5 mL centrifuge tubes, and store at -80 °C.

[0034] Further, the specific steps of step S5 include:

[0035] S501: Take 100 μL of competent cells, mix them with 5 μg of P1600 - GENT - RS15945 - FLAG plasmid, add them to a pre-cooled 0.2 cm electroporation cuvette, and place it on ice for 5 minutes.

[0036] S502: Perform electroporation according to the electroporator parameters, with a high-voltage pulse of 2.5 kV / cm, a capacitance of 25 μF, a resistance of 200 Ω, and an electric shock for 5 ms.

[0037] S503: Immediately add 500 μL of LB solution for elution after electroporation, transfer it into a 1.5 mL centrifuge tube, and resuscitate at 37 °C and 250 rpm for 1 hour.

[0038] S504: Spread on an LB screening plate containing 25 μg / mL Gm and incubate at 37 °C for 48 hours until obvious colonies grow.

[0039] S505: Pick a single colony into an LB liquid medium containing 25 μg / mL Gm and culture at 37 °C for 24 hours for subsequent PCR identification.

[0040] The present invention also provides the application of the ncd2 gene overexpression strain in the histological structure and flora of the broiler intestine.

[0041] The present invention has the following beneficial effects compared with the prior art:

[0042] 1. For the ncd2 gene overexpression strain, the overexpression of the ncd2 gene can enhance the ability of Sphingomonas to utilize specific substrates, improve its activity in metabolic processes such as ammonia degradation. Overexpressing the ncd2 gene may enable the strain to exhibit stronger viability and adaptability under adverse environmental conditions (such as high concentrations of ammonia or other inhibitory substances), that is, increase stress resistance. The genetically engineered strain can play an important role in the fields of bioremediation and environmental governance, especially in the treatment of ammonia pollution.

[0043] 2. For its construction method, it has an efficient screening mechanism, that is, using a resistance marker gene to screen successfully transformed strains, which can improve the screening efficiency and ensure the success rate of the experiment. Using modern molecular cloning techniques (such as Gibson assembly), it can efficiently and accurately construct vectors containing the target gene, reduce operation steps and time. Combining methods such as electroporation and heat shock can improve the cell transformation efficiency and obtain more transformants, and at the same time has high repeatability, suitable for large-scale experiments and research.

[0044] 3. After application to broilers, adding 4×10 5 CFU / mL of different strains of Sphingomonas in the drinking water of broilers can improve the composition of the intestinal microflora and the histological structure of the intestine. At the same time, it can significantly reduce the contents of total nitrogen, uric acid, and ammonium nitrogen in the chicken manure of broilers, while significantly increasing the content of nitrate nitrogen, and can increase the slaughter rate, thereby increasing economic benefits. Description of the Drawings

[0045] Figure 1 It is a schematic diagram of the ncd2 gene containing homologous arms amplified by PCR in the present invention.

[0046] Figure 2Schematic diagram of the ncd2 gene of the recombinant plasmid amplified by colony PCR in the present invention.

[0047] Figure 3 Schematic diagram of the overexpression of the ncd2 gene in Sphingomonas verified by PCR amplification in the present invention.

[0048] Figure 4 Schematic diagram of the comparison of the results of real-time fluorescence quantitative PCR detection in the present invention.

[0049] Figure 5 Schematic diagram of the construction targeting fragment of the upstream homologous recombination arm, downstream homologous recombination arm and gentamicin resistance gene in the present invention.

[0050] Figure 6 Schematic diagram of the result of the construction targeting fragment of the upstream homologous recombination arm, downstream homologous recombination arm and gentamicin resistance gene in the present invention.

[0051] Figure 7 Schematic diagram of the PCR amplification using the internal primer of the ncd2 gene in the present invention.

[0052] Figure 8 Schematic diagram of the result after PCR amplification using the internal primer of the ncd2 gene in the present invention. Detailed implementation manners

[0053] To facilitate the understanding of the present invention, the device of the present invention will be described more comprehensively below with reference to the relevant drawings. Embodiments of the device are shown in the drawings. However, the device can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, these embodiments are provided to make the disclosure of the present invention more thorough and comprehensive.

[0054] In the description of the present invention, it should be noted that, unless otherwise clearly defined and limited, the terms "installation", "connection", and "setting" should be understood in a broad sense. For example, it can be fixedly connected and set, or detachably connected and set, or integrally connected and set. For those of ordinary skill in the art, the specific meanings of the above terms in the present invention can be understood according to specific situations.

[0055] Example 1

[0056] This example provides a method for constructing an overexpression strain of the ncd2 gene in Sphingomonas. The overexpression strain of the ncd2 gene is obtained by introducing the ncd2 gene into Sphingomonas through a recombinant plasmid or integrating it into the chromosome of Sphingomonas by genetic engineering means;

[0057] The Sphingomonas sp. is Sphingomonas sp. Z392, which is provided by the Fermentation Engineering Laboratory of the School of Biology and Food Engineering, Huanghuai University.

[0058] Example 2

[0059] A method for constructing an overexpression strain of the ncd2 gene of Sphingomonas sp. comprises the following steps:

[0060] S1. Determine the resistance characteristics of Sphingomonas sp. by measuring the minimum inhibitory concentration with appropriate antibiotics; S101: Select gentamicin, streptomycin, and tetracycline to measure the minimum inhibitory concentration of Sphingomonas sp.;

[0061] S102: Inoculate a certain volume of Sphingomonas sp. into each well and incubate overnight at 37°C;

[0062] S103: Measure the absorbance value of each well at a wavelength of 600 nm using a spectrophotometer.

[0063] The reagents are shown in the following table:

[0064]

[0065]

[0066] S2. First, extract genomic DNA from Sphingomonas sp. and amplify the ncd2 gene using specific primers. Subsequently, perform Gibson assembly with the linearized vector to construct a recombinant plasmid;

[0067] S201: Extract the genomic DNA of Sphingomonas sp., and amplify the ncd2 gene fragment containing the P1600-gent-Flag homologous arm using the RBSF and pFLP3-RRNBTR primers. The amplification system is shown in the following table;

[0068]

[0069] S202: Perform gel electrophoresis on the amplification product and use an agarose gel recovery kit to recover the target product;

[0070] S203: Digest the p1600-gent-Flag vector backbone with NcoI and EcoRI, and incubate at 37°C for 1 hour to linearize the vector. The digestion system is as follows;

[0071]

[0072] S204: Recombine the linearized vector and the ncd2 gene fragment containing the homologous arm using Gibson assembly, and incubate at 50°C for 30 minutes. The assembly system is shown in the following table.

[0073]

[0074] S3. Transform the recombinant plasmid into competent cells, and through heat shock and recovery culture, make it grow on the selective medium, and finally screen out the successfully transformed strains;

[0075] S301: Thaw DH5α competent cells on ice, add 10 μL of the recombinant product to 100 μL of competent cells, flick the tube wall gently to mix evenly, and let it stand on ice for 30 minutes;

[0076] S302: Perform heat shock in a 42 °C water bath for 45 seconds, and immediately place it on ice to cool for 2 - 3 minutes;

[0077] S303: Add LB medium (without antibiotics), shake the bacteria at 37 °C for 1 hour, and the rotation speed is 200 - 250 rpm;

[0078] S304: Preheat the LB solid medium plate containing 50 μg / mL gentamicin in a 30 °C incubator;

[0079] S305: Centrifuge at 5000 rpm for 5 minutes, discard 900 μL of the supernatant, resuspend the bacterial cells and spread them evenly on the plate containing 50 μg / mL Gm, and incubate them upside down in a 37 °C incubator for 12 - 16 hours;

[0080] S306: Pick monoclonal colonies on the recombinant reaction transformation plate for colony PCR identification.

[0081] It should be noted that the process of the recombinant plasmid containing the ncd2 fragment is as follows: Gel extraction and recovery of the ncd2 gene with homologous arms are assembled with the linearized P1600 - gent - Flag plasmid by Gibson assembly, and then transformed into DH5α competent cells to obtain positive strains. The results of colony PCR verification show that the recombinant plasmid contains the ncd2 gene, as Figure 2 shown, and gel extraction and recovery followed by sequencing verify the successful construction of the recombinant plasmid, where M: DL6000 DNA Marker, unit is bp; 1 and 2: ncd2 fragments.

[0082] S4. Culture Sphingomonas, prepare competent cells, transform the plasmid into these cells, and then screen out the successfully transformed clones on the selective medium;

[0083] S401: Inoculate a single colony from a freshly cultured plate into 3 mL of LB liquid medium, culture at 200 rpm and 37 °C for 16 hours;

[0084] S402: Inoculate 1.5 mL of overnight culture into 150 mL of fresh LB medium (contained in a 500 mL Erlenmeyer flask), and culture at 37 °C with 200 rpm until the cell number reaches approximately 1×10 8 CFU / mL;

[0085] S403: Transfer the bacterial solution to a 50 mL centrifuge tube, ice-bath for 15 minutes, then centrifuge at 4 °C, 4500 rpm for 5 minutes to collect the cells;

[0086] S404: Discard the supernatant, resuspend the cells with 30 mL of ice-cold 10% glycerol, ice-bath for 5 minutes, then centrifuge at 4 °C, 4500 rpm for 5 minutes to collect the cells;

[0087] S405: Resuspend the cells with 2 mL of pre-cooled 10% glycerol, with a cell concentration of 1×10^10 CFU / mL, aliquot into 1.5 mL centrifuge tubes, and store at -80 °C.

[0088] S5: Extract genomic DNA from the transformed strain and perform PCR verification to confirm the successful overexpression of the ncd2 gene, i.e., the electrotransformation of Sphingomonas;

[0089] S501: Take 100 μL of competent cells, mix with 5 μg of P1600-GENT-RS15945-FLAG plasmid, then add to a pre-cooled 0.2 cm electroporation cuvette (electrode gap), and place on ice for 5 minutes.

[0090] S502: Perform electrotransformation according to the electroporator parameters, with a high-voltage pulse of 2.5 kV / cm, a capacitance of 25 μF, a resistance of 200 Ω, and an electric shock for 5 ms.

[0091] S503: Immediately add 500 μL of LB solution for elution after electroporation, transfer to a 1.5 mL centrifuge tube, and resuscitate at 37 °C with 250 rpm for 1 hour.

[0092] S504: Spread on an LB screening plate containing 25 μg / mL Gm, and culture at 37 °C for 48 hours until obvious colonies grow.

[0093] S505: Pick a single colony into an LB liquid medium containing 25 μg / mL Gm, and culture at 37 °C for 24 hours for subsequent PCR identification.

[0094] S6: Optimize the culture conditions in the obtained ncd2 overexpressing strain.

[0095] It should be noted that in order to achieve precise gene knockout, ensure the effective integration of the reintroduced genes, and improve the screening efficiency, the ncd2 fragment containing homologous arms can be amplified. That is, the ncd2 gene fragment containing the P1600-gent-Flag homologous arms is amplified using the RBSF and pFLP3-RRNBTR primers, including the ncd2 gene fragment (984bp), a 23bp upstream arm, and a 68bp downstream arm, totaling 1075bp. As Figure 1 shown, where M: DL6000 DNA Marker, with the unit of bp; 1: ncd2 fragment containing homologous arms.

[0096] As Figure 3 shown, Sphingomonas overexpressing ncd2 is cultured in a resistant LB liquid medium. Genomic DNA is extracted using a bacterial genomic DNA extraction kit. After PCR amplification using primers containing partial plasmid genes, the size of the PCR-amplified fragment is approximately 1069bp. This fragment contains the ncd2 fragment and partial fragments on the plasmid. Then, gel extraction and sequencing verification are successful. Where M: DL6000 DNA Marker, with the unit of bp; 1-10: ncd2 fragment.

[0097] In addition, to further verify the expression level of this gene in the ncd2 overexpressing strain, real-time fluorescence quantitative PCR technology is used to detect the ncd2 expression in the ncd2 overexpressing strain. The results are as Figure 4 shown, and the results prove that the expression level of the ncd2 gene in the overexpressing strain is significantly higher than that in the wild strain.

[0098] Example 3

[0099] The method for constructing an ncd2 gene knockout strain includes the following steps:

[0100] S1. By introducing a resistance marker, the successfully transformed or knocked-out strains are screened.

[0101] S101. First, gentamicin, streptomycin, and tetracycline are selected to determine the minimum inhibitory concentration (MIC) of Sphingomonas to determine its sensitivity to different antibiotics.

[0102] S102. Then, the upstream and downstream homologous recombination arms of the ncd2 gene are amplified using the ultra-fidelity enzyme PrimeSTAR Max (TaKaRa), and the pJQ200SK plasmid is extracted from Escherichia coli to amplify the fragment containing gentamicin resistance.

[0103] S103. Finally, the upstream homologous recombination arm, the gentamicin resistance gene fragment, and the downstream homologous recombination arm were fused using fusion PCR technology to obtain the complete targeting fragment Δncd2::Gm, which was then cloned into the suicide plasmid pCVD442 to obtain the targeting plasmid pCVD442-Δncd2::Gm.

[0104] The amplification results of the upstream homologous recombination arm (828 bp), the downstream homologous recombination arm (829 bp), and the gentamicin resistance gene (936 bp) are as Figure 5 shown, where 1 represents the upstream homologous recombination arm, 2 represents the downstream homologous recombination arm, and 3 represents the gentamicin resistance gene;

[0105] After electrophoresis, these three fragments were excised and recovered, and fusion PCR was performed. The length of the obtained targeting sequence (upstream homologous recombination arm - gentamicin resistance gene coding sequence - downstream homologous recombination arm) was 2546 bp, as Figure 6 shown, where 1 represents the targeting fragment of the upstream homologous recombination arm - Gm resistance gene coding sequence - downstream homologous recombination arm with a length of 2546 bp; M represents DL6000 DNA Marker, with the unit of bp.

[0106] S2. Preparation of the donor bacterium;

[0107] S201. The targeting vector pCVD442-Δncd2::Gm was electrotransformed into the E. coli β2155 strain;

[0108] S202. Then, the electrotransformed liquid was spread on an LB plate containing sodium ampicillin and diaminopimelic acid, and cultured at 37°C until monoclonal formation;

[0109] S203. Finally, it was confirmed that the formed monoclonal was the donor strain β2155 / pCVD442-Ancd2::Gm;

[0110] S3. Conjugation experiment and knockout of the ncd2 gene;

[0111] S301. The recipient bacterium Sphingomonas was streaked on a TSB plate and cultured at 37°C until monoclonal formation;

[0112] S302. Then, a monoclonal was picked and inoculated into 3 mL of TSB medium, and cultured overnight at 37°C and 220 rpm;

[0113] S303. Then, a monoclonal of the donor bacterium β2155 / pCVD442-Ancd2::Gm was picked and inoculated into 3 mL of TSB (containing 100 μg / mL Amp and 0.5 mM DAP), and cultured overnight at 37°C and 220 rpm;

[0114] S304. Mix 500 μL of donor bacterial solution with 500 μL of recipient bacterial solution for a conjugation experiment. Take 50 μL of the post-conjugation bacterial solution and spread it on a TSB plate containing Gm (33 μg / mL), and culture it at 30 °C until monoclonal formation;

[0115] S305. Randomly select 10 clones on the resistance plate, pick them into the same tube of 100 μL of TSB medium, mix well, then take 10 μL and inoculate it into 3 mL of TSB medium, and culture it overnight at 30 °C and 220 rpm;

[0116] S306. The next day, take 50 μL of the culture solution and spread it on an LB plate containing 10% sucrose (containing 33 μg / mL of Gm and no NaCl), place it at 30 °C and culture until monoclonal formation. Randomly select 14 monoclonal colonies and inoculate them into 3 mL of TSB medium (containing 33 μg / mL of Gm) for PCR identification;

[0117] S307. Take a small amount of the bacterial solution of the 1st clone and inoculate it into 3 mL of TSB (containing 33 μg / mL of Gm), culture it overnight at 37 °C and 220 rpm. The next day, take a small amount of the bacterial solution and perform a PCR reaction with the outer primers to verify whether the ncd2 gene has been successfully knocked out;

[0118] S4. Real-time fluorescence quantitative PCR;

[0119] S401. Culture the ncd2 overexpression strain and the wild strain respectively, collect the bacterial cells and quickly freeze them in liquid nitrogen;

[0120] S402. Then, use the Bacteria RNA Extraction Kit to extract the total RNA of bacteria, and use 4×gDNA Wiper Mix to remove genomic DNA;

[0121] S403. Reverse transcribe it into cDNA using 5×HiScript III RT SuperMix, and use SYBR Green Master Mix for fluorescence quantitative PCR detection of the ncd2 gene, with the 16s conserved sequence as the internal reference gene;

[0122] S5. Optimization of culture conditions;

[0123] S501. Based on LB medium, use the growth spectrum method to explore the nutritional requirements of different Sphingomonas strains, and determine the optimal carbon source, nitrogen source and their contents;

[0124] S502. Determine the optimal culture temperature, initial pH value, inoculation amount and shaking frequency of the shaker for the 3 strains through single-factor experiments;

[0125] S503. According to the results of single-factor experiments, use the SPSSAU orthogonal experimental design system and adopt the two-factor four-level orthogonal design method to determine the optimal culture conditions;

[0126] S6. Determination of the growth curve of the strain and the total number of bacteria in the stationary phase;

[0127] S601. Cultivate the 3 strains according to the optimized culture medium and conditions, sample every 2 hours, measure the OD value at a wavelength of 600 nm, and plot the relationship curve between OD600 and the culture time;

[0128] S602. For the total number of bacteria in the stationary phase, use the plate counting method for statistics.

[0129] Culture the Sphingomonas ncd2 knockout strain and the original Sphingomonas in TSB liquid medium. Take a small amount of the above culture solutions respectively and perform PCR amplification using the internal primers of the ncd2 gene. The amplification results are as Figure 7 shown. The results show that the 5th, 7th, and 10th clones are positive amplifications, and the 1st - 4th, 6th, 8th, 9th, and 11th - 14th clones are all negative amplifications. Thus, it is speculated that the gene ncd2 may have been knocked out, but PCR amplification with outer primers is still required for further identification;

[0130] Perform PCR reaction on the culture solutions corresponding to the negative amplifications using the outer primers. The results are as Figure 8 shown: The length of the specifically amplified product fragment is consistent with that of the fragment of the replaced strain (2686 bp). The PCR amplification product was sent for sequencing. The results show that the gentamicin resistance gene has replaced the original gene at the target position, which is consistent with the design scheme. Thus, it is shown that this clone is the ncd2 gene knockout clone.

[0131] Example 4

[0132] The application of the Sphingomonas ncd2 gene overexpression strain to the histological structure of the broiler intestine. Add 4×10 5 CFU / mL of different strains of Sphingomonas to the drinking water of broilers, which can improve the composition of the intestinal microbial flora and the histological structure of the intestine. Moreover, the degree of improvement of the overexpression strain is better than that of the wild strain, and the wild strain is better than the gene knockout strain. However, the relative abundances of Faecalibacterium and Lactobacillus intestinalis in the ileum of broilers, and the relative abundances of Lachnospiraceae, Faecalibacterium, Ruminococcus, and Odoribacter in the cecum decrease, while the relative abundances of Clostridium in the duodenum, Shigella in the ileum, and Staphylococcus in the jejunum increase, which is likely to cause the occurrence of digestive tract diseases. Therefore, when in use, other microorganisms need to be used in combination to inhibit the reproduction of potentially harmful bacteria and ensure the health and breeding benefits of broilers.

[0133] Example 5

[0134] The ammonia reduction ability of the Sphingomonas ncd2 gene overexpression strain is applied to the growth of broilers. After adding 4×10 5 CFU / mL of the Sphingomonas ncd2 gene overexpression strain to the drinking water, the contents of total nitrogen, uric acid, and ammonium nitrogen in the chicken manure of broilers can be significantly decreased (p < 0.05), while the content of nitrate nitrogen is significantly increased (p < 0.05). The ammonia reduction effect of the ncd2 gene overexpression strain is the most significant. Compared with the control group, the ammonia concentration in the chicken house at 42 days of age can be decreased by 43.33%, and compared with the wild strain, it is decreased by 14.17% (p < 0.05), and it is decreased by 25.83% more than the ncd2 gene knockout strain. Compared with the control group, the slaughter weight can be increased by 8.66%, and the European index can be increased by 17.76%. Compared with the wild strain, the slaughter weight can be increased by 3.77%, and the European index can be increased by 5.14%. Compared with the ncd2 gene knockout strain, the slaughter weight can be increased by 6.59%, and the European index can be increased by 10.09%, enabling broilers to obtain better economic benefits.

[0135] It should be noted that the structure described in the present invention can be implemented in many different forms and is not limited to the embodiments. Any equivalent transformation made by those of ordinary skill in the art using the specification and drawings of the present invention, or directly or indirectly applied in other related technical fields, such as the loading and unloading of other articles, is included in the protection scope of the present invention.

Claims

1. A Sphingomonas ncd2 gene overexpression strain, characterized in that: It is named as the Sphingomonas ncd2 gene overexpression strain. The Sphingomonas ncd2 gene overexpression strain is deposited in the China Center for Type Culture Collection. The preservation date is March 18, 2025, and the preservation number is CGMCC No.33862.

2. A method for constructing a sphingomonas ncd2 gene overexpression strain, characterized in that: The following steps are involved: S1. Determine the resistance characteristics of Sphingomonas by selecting appropriate antibiotics for minimum inhibitory concentration determination; S2, first extract genomic DNA from Sphingomonas, and use specific primers to amplify the ncd2 gene, and then perform Gibson assembly with the linearized vector to construct a recombinant plasmid; S3, transforming the recombinant plasmid into competent cells, growing them on selective medium through heat shock and recovery culture, and finally screening out the successfully transformed strains; S4, culturing Sphingomonas to prepare competent cells, transforming the plasmid into these cells, and then screening the successfully transformed clones on a selective medium; S5. Extract genomic DNA from the transformed strain and perform PCR verification to confirm the successful overexpression of the ncd2 gene; S6. Optimize the culture conditions in the obtained ncd2 overexpression strain.

3. The method for constructing the ncd2 gene overexpression strain of Sphingomonas as claimed in claim 2, characterized in that: The step S1 specifically includes: S101: Gentomycin, streptomycin and tetracycline were selected to determine the minimum inhibitory concentration of Sphingomonas; S102: Each well was inoculated with a certain volume of Sphingomonas and cultured overnight at 37°C; S103: Measure the absorbance value of each well at a wavelength of 600 nm using a spectrophotometer.

4. The method for constructing a sphingomonas ncd2 gene overexpression strain as claimed in claim 2, characterized in that: The step S2 specifically includes: S201: Extract genomic DNA of Sphingomonas and use RBSF and pFLP3-RRNBTR primers to amplify the ncd2 gene fragment containing the P1600-gent-Flag homology arm; S202: performing gel electrophoresis on the amplified product, and recovering the target product using an agarose gel recovery kit; S203: digest the p1600-gent-Flag vector backbone with NcoI and EcoRI and incubate at 37°C for 1 hour to linearize the vector; S204: Recombine the linearized vector and the ncd2 gene fragment containing the homology arms using Gibson assembly and incubate at 50°C for 30 minutes.

5. The method for constructing a Sphingomonas ncd2 gene overexpression strain as claimed in claim 2, characterized in that: The step S3 specifically includes: S301: Thaw DH5α competent cells on ice, add 10 μL of the recombinant product to 100 μL of competent cells, flick the tube wall to mix, and let stand on ice for 30 minutes; S302: Heat shock in a 42°C water bath for 45 seconds, then immediately cool on ice for 2-3 minutes; S303: Add LB medium without adding antibiotics, shake the culture at 37°C for 1 hour at a speed of 200-250 rpm; S304: Preheat LB solid medium plates containing 50 μg / mL gentamicin in a 30°C incubator; S305: Centrifuge at 5000 rpm for 5 minutes, discard 900 μL of supernatant, resuspend the cells and gently spread them on a plate containing 50 μg / mL Gm, and incubate inverted in a 37°C incubator for 12-16 hours; S306: Pick a single clone on the recombination reaction transformation plate for colony PCR identification.

6. The method for constructing a Sphingomonas ncd2 gene overexpression strain as claimed in claim 2, characterized in that: The step S4 specifically includes: S401: Inoculate a single colony from a freshly cultured plate into 3 mL of LB liquid medium and culture at 200 rpm and 37°C for 16 hours; S402: Inoculate 1.5 mL of overnight cultured bacterial solution into 150 mL of fresh LB medium and culture at 200 rpm and 37°C until the bacterial count reaches about 1×10 8 CFU / mL; S403: Transfer the bacterial solution to a 50 mL centrifuge tube, place on ice for 15 minutes, and then centrifuge at 4°C, 4500 rpm, for 5 minutes to collect the bacteria; S404: discard the supernatant, resuspend the cells with 30 mL of ice-cold 10% glycerol, place on ice for 5 minutes, then centrifuge at 4°C, 4500 rpm, for 5 minutes to collect the cells; S405: Resuspend the cells in 2 mL of pre-cooled 10% glycerol to a cell concentration of 1 × 10 10 CFU / mL, divided into 1.5mL centrifuge tubes and stored at -80℃.

7. The method for constructing a sphingomonas ncd2 gene overexpression strain according to claim 2, characterized in that: The step S5 specifically includes: S501: Take 100 μL of competent cells, mix with 5 μg of P1600-GENT-RS15945-FLAG plasmid, add to a pre-cooled 0.2 cm electroporation transformation cup, and place on ice for 5 minutes. S502: Perform electroporation according to the parameters of the electroporator: high voltage pulse 2.5 kV / cm, capacitance 25 μF, resistance 200 Ω, and electric shock 5 ms. S503: Immediately after electroporation, add 500 μL LB solution for elution, add to a 1.5 mL centrifuge tube, and resuscitate at 37°C, 250 rpm for 1 hour. S504: Spread LB screening plates containing 25 μg / mL Gm and culture at 37°C for 48 hours until obvious clones grow. S505: Pick a single clone into LB liquid medium containing 25 μg / mL Gm and culture at 37°C for 24 hours for subsequent PCR identification.

8. Application of the ncd2 gene overexpression strain according to claim 1, or the ncd2 gene overexpression strain constructed according to any one of claims 2-7 in the histological structure and flora of the broiler intestine.

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