CN113248582A - Transformation and regulation method of nitrogen-fixing microorganisms - Google Patents

Transformation and regulation method of nitrogen-fixing microorganisms Download PDF

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CN113248582A
CN113248582A CN202110507967.9A CN202110507967A CN113248582A CN 113248582 A CN113248582 A CN 113248582A CN 202110507967 A CN202110507967 A CN 202110507967A CN 113248582 A CN113248582 A CN 113248582A
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bacteria
nitrogen
protein
methanotrophic
growth
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费强
张天晴
郭树奇
傅容湛
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Xian Jiaotong University
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Xian Jiaotong University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor

Abstract

The invention discloses a method for modifying and regulating nitrogen-fixing microorganisms. The invention provides a method for determining whether a test protein in methanotrophic bacteria participates in the bacterial growth of methanotrophic bacteria by using nitrogen as a nitrogen source, which comprises the following steps: taking methanotrophic bacteria as spawn, and knocking out coding genes of tested proteins in the spawn through homologous recombination to obtain knock-out bacteria; parallel culture; if the time of entering the decay period of the growth curve of the knock-out bacteria is earlier than that of the emerging bacteria, the tested protein is a positive regulation protein participating in the bacterial quantity growth of the methanotrophic bacteria by using nitrogen as a nitrogen source; if the time of the growth curve of the knockout bacteria entering the decay period is the same as or later than that of the outgrowth bacteria and the mass peak value of the outgrowth bacteria is higher than that of the outgrowth bacteria, the tested protein is a negative regulation protein which participates in the growth of the methanotrophic bacteria by using nitrogen as a nitrogen source.

Description

Transformation and regulation method of nitrogen-fixing microorganisms
Technical Field
The invention belongs to the technical field of biology, and relates to a method for modifying and regulating nitrogen-fixing microorganisms.
Background
Biological nitrogen fixation is an important part of the global nitrogen cycle, as it supplements the total nitrogen content of the biosphere and compensates for losses due to denitrification. Therefore, the research on the nitrogen fixation mechanism in the nitrogen fixation microorganisms is beneficial to understanding the action rule of global nitrogen circulation.
The ability to fix nitrogen has now been found in most phylogenetic groups of bacteria, including methanotrophic bacteria that are capable of utilizing methane as the sole carbon source, and studies of the intersection between carbon and nitrogen metabolism of methanotrophic bacteria have helped expand our opinion that methanotrophic bacteria influence the biogeochemical cycle.
Although nitrogenase is a protein that is well conserved in structure and function, the regulation pattern of its associated transcription factors varies greatly among different nitrogen-fixing microorganisms. The methanotrophic bacteria have a complete nitrogen fixation metabolism system in the cell, but few technical methods for stimulating the nitrogen fixation metabolism in the methanotrophic bacteria are developed. In addition, at present, relatively few reports are reported for nitrogen fixation metabolic mechanisms in methanotrophic bacteria, and particularly, the research on the nitrogen fixation gene transcription factor in methanotrophic bacteria is deficient.
In recent years, genetic tools such as gene knockout gradually establish a corresponding system in methanotrophic bacteria, but most of the genetic tools use a mode of constructing plasmids in vitro to knock out target genes, wherein a genetic operation means of parental combination or even parental combination is possibly involved to introduce a vector into a target strain, and the complexity of genetic operation increases the experimental difficulty and simultaneously reduces the high efficiency of plasmid introduction.
Disclosure of Invention
The invention aims to provide a method for modifying and regulating nitrogen-fixing microorganisms.
The invention provides a method for determining whether a test protein in methanotrophic bacteria participates in the bacterial growth of methanotrophic bacteria by using nitrogen as a nitrogen source, which comprises the following steps:
taking methanotrophic bacteria as spawn, and knocking out coding genes of tested proteins in the spawn through homologous recombination to obtain knock-out bacteria;
respectively carrying out parallel culture on the spawn running bacteria and the knockout bacteria; if the time of entering the decay period of the growth curve of the knock-out bacteria is earlier than that of the emerging bacteria, the tested protein is a positive regulation protein participating in the bacterial quantity growth of the methanotrophic bacteria by using nitrogen as a nitrogen source; if the time of the growth curve of the knock-out bacteria entering the decay phase is the same as or later than that of the outgrowth bacteria and the mass peak value of the growth curve is higher than that of the outgrowth bacteria, the tested protein is a negative regulation protein participating in the growth of the methanotrophic bacteria by using nitrogen as a nitrogen source;
the culture method comprises the following steps: culturing in a sealed environment by adopting a specific culture medium; the gas in the sealed environment is a mixed gas of nitrogen and oxygen, and the volume percentage of the oxygen in the mixed gas is 15-18%; the only nitrogen source in the specific medium is nitrate ion, and its concentration in the medium (initial concentration) is 0.25-0.5 g.L-1。
In the method, after a nitrogen source in a culture medium is exhausted, if the time of entering a decay period of a growth curve of knock-out bacteria is earlier than that of spawn running, a test protein is a positive control protein participating in the growth of the methanotrophic bacteria by using nitrogen as the nitrogen source; if the time of the growth curve of the knockout bacteria entering the decay period is the same as or later than that of the outgrowth bacteria and the mass peak value of the outgrowth bacteria is higher than that of the outgrowth bacteria, the tested protein is a negative regulation protein which participates in the growth of the methanotrophic bacteria by using nitrogen as a nitrogen source.
Specifically, the concentration of nitrate ions in the medium (initial concentration) was 0.31 g.L-1。
Specifically, the nitrate ion is KNO3Providing KNO3The concentration in the medium (initial concentration) was 0.5 g.L-1。
Specifically, the volume percentage of oxygen in the mixed gas is 17.85%.
The exhaustion of the nitrogen source in the medium means that the concentration of nitrate ions in the medium reaches a low value.
The invention also provides a method for determining whether the test protein in the methanotrophic bacterium participates in the bacterial growth of the methanotrophic bacterium by using nitrogen as a nitrogen source, which comprises the following steps:
taking methanotrophic bacteria as spawn, and knocking out coding genes of tested proteins in the spawn through homologous recombination to obtain knock-out bacteria;
respectively carrying out parallel culture on the spawn running bacteria and the knockout bacteria; if the time of entering the decay period of the growth curve of the knock-out bacteria is earlier than that of the emerging bacteria, the tested protein is a positive regulation protein participating in the bacterial quantity growth of the methanotrophic bacteria by using nitrogen as a nitrogen source;
the culture method comprises the following steps: culturing in a sealed environment by adopting a specific culture medium; the gas in the sealed environment is a mixed gas of nitrogen and oxygen, and the volume percentage of the oxygen in the mixed gas is 15-18%; the only nitrogen source in the specific medium is nitrate ion, and its concentration in the medium (initial concentration) is 0.25-0.5 g.L-1。
In the method, after the nitrogen source in the culture medium is exhausted, if the time of entering the death phase of the growth curve of the knock-out bacteria is earlier than that of the emerging bacteria, the protein to be tested is positive control protein which participates in the growth of the methanotrophic bacteria by using nitrogen as the nitrogen source.
Specifically, the concentration of nitrate ions in the medium (initial concentration) was 0.31 g.L-1。
Specifically, the nitrate ion is KNO3Providing KNO3The concentration in the medium (initial concentration) was 0.5 g.L-1。
Specifically, the volume percentage of oxygen in the mixed gas is 17.85%.
The exhaustion of the nitrogen source in the medium means that the concentration of nitrate ions in the medium reaches a low value.
The invention also provides a method for determining whether the test protein in the methanotrophic bacterium participates in the bacterial growth of the methanotrophic bacterium by using nitrogen as a nitrogen source, which comprises the following steps:
taking methanotrophic bacteria as spawn, and knocking out coding genes of tested proteins in the spawn through homologous recombination to obtain knock-out bacteria;
respectively carrying out parallel culture on the spawn running bacteria and the knockout bacteria; if the time of the growth curve of the knock-out bacteria entering the decay phase is the same as or later than that of the outgrowth bacteria and the mass peak value of the growth curve is higher than that of the outgrowth bacteria, the tested protein is a negative regulation protein participating in the growth of the methanotrophic bacteria by using nitrogen as a nitrogen source;
the method of culturingComprises the following steps: culturing in a sealed environment by adopting a specific culture medium; the gas in the sealed environment is a mixed gas of nitrogen and oxygen, and the volume percentage of the oxygen in the mixed gas is 15-18%; the only nitrogen source in the specific medium is nitrate ion, and its concentration in the medium (initial concentration) is 0.25-0.5 g.L-1。
In the method, after the nitrogen source in the culture medium is exhausted, if the time of the growth curve of the knock-out bacteria entering the death phase is the same as or later than that of the outgrowth bacteria and the mass peak value of the outgrowth bacteria is higher than that of the outgrowth bacteria, the protein to be tested is negative regulation protein which participates in the growth of the methanotrophic bacteria by using nitrogen as the nitrogen source.
Specifically, the concentration of nitrate ions in the medium (initial concentration) was 0.31 g.L-1。
Specifically, the nitrate ion is KNO3Providing KNO3The concentration in the medium (initial concentration) was 0.5 g.L-1。
Specifically, the volume percentage of oxygen in the mixed gas is 17.85%.
The exhaustion of the nitrogen source in the medium means that the concentration of nitrate ions in the medium reaches a low value.
Specifically, in any of the above methods, the initial OD of the system at the time of initiation of the culture600nmThe value was 0.5.
Specifically, in any of the above methods, the time for the growth curve of the starting bacterium to enter the death phase may be from 84 to 108 hours of culture, and specifically may be from 84 to 96 hours of culture.
Specifically, in any of the above methods, the time for the growth curve of the knockout bacterium to enter the death phase may be within 84 hours of culture, and specifically may be 48 to 72 hours of culture.
Specifically, in any of the above methods, the time for the growth curve of the knockout bacterium to enter the death phase may be 84 to 108 hours of culture.
The invention also provides a method for determining whether the test protein in the methanotrophic bacterium participates in the bacterial growth of the methanotrophic bacterium by using nitrogen as a nitrogen source, which comprises the following steps:
taking methanotrophic bacteria as spawn, and knocking out coding genes of tested proteins in the spawn through homologous recombination to obtain knock-out bacteria;
culturing the knockout bacterium; if the growth curve of the knock-out bacteria enters a decline period within 72 hours of culture, the tested protein is a positive regulation protein participating in the growth of the methanotrophic bacteria by using nitrogen as a nitrogen source;
the culture method comprises the following steps: culturing in a sealed environment by adopting a specific culture medium; the gas in the sealed environment is a mixed gas of nitrogen and oxygen, and the volume percentage of the oxygen in the mixed gas is 15-18%; the only nitrogen source in the specific medium is nitrate ion, and its concentration in the medium (initial concentration) is 0.25-0.5 g.L-1。
The time period of 72 hours or less may be 48 hours or less.
The time period within 72 hours may be specifically 48 to 72 hours.
Specifically, the concentration of nitrate ions in the medium (initial concentration) was 0.31 g.L-1。
Specifically, the nitrate ion is KNO3Providing KNO3The concentration in the medium (initial concentration) was 0.5 g.L-1。
Specifically, the volume percentage of oxygen in the mixed gas is 17.85%.
Specifically, in any of the above methods, the initial OD of the system at the time of initiation of the culture600nmThe value was 0.5.
Specifically, in any of the above methods, the culturing method may be: the sterilized injection bottle (500mL capacity) is filled with 100mL of sterilized liquid hNMS culture medium, and then the test bacteria is inoculated, the initial OD of the system600nmA value of 0.5; then vacuumizing the injection bottle, introducing a mixed gas of nitrogen and oxygen (the volume percentage of the oxygen in the mixed gas is 17.85%) to normal air pressure, and then sealing; then, the cells were cultured at 30 ℃ for 108 hours with shaking at 200 rpm. During the culture process, the following steps are carried out again every 12 hours: vacuumizing the injection bottle, and introducing a mixed gas of nitrogen and oxygen (mixed gas)Wherein the volume percentage of oxygen is 17.85%) to normal pressure, and then sealing.
The invention also provides a methanotrophic bacterium transformation method, which comprises the following steps:
determining whether the test protein in the methanotrophic bacteria participates in the growth of the methanotrophic bacteria by using nitrogen as a nitrogen source;
and carrying out genetic engineering modification on the methanotrophic bacteria according to whether the test protein in the methanotrophic bacteria participates in the growth of the methanotrophic bacteria by using nitrogen as a nitrogen source.
The genetic engineering is modified to knock out the coding gene of the test protein in the methanotrophic bacterium.
The genetic engineering is modified to knock out the encoding gene of the test protein in the methanotrophic bacterium through homologous recombination.
If the methanotrophic bacterium test protein is a positive regulation protein participating in the growth of the methanotrophic bacterium by using nitrogen as a nitrogen source, the engineering bacterium with the suppressed growth of the methanotrophic bacterium by using nitrogen as a nitrogen source is obtained by genetic engineering transformation.
If the methanotrophic bacterium test protein is a negative control protein participating in the growth of the methanotrophic bacterium by using nitrogen as a nitrogen source, the engineering bacterium which is promoted to grow the amount by using nitrogen as the nitrogen source is obtained by genetic engineering transformation.
The low amount value may be 99 mg.L-1The following.
The low amount value may be 70 mg.L-1The following.
The low amount value may be 60 mg.L-1The following.
The low amount value may be 50 mg.L-1The following.
The low amount value may be 40 mg.L-1The following.
The low amount value may be 30 mg.L-1The following.
The low amount value may be 20 mg.L-1The following.
In any of the above methods, the knockout of the gene encoding the test protein in the producing strain (methanotrophic bacterium) by homologous recombination is carried out by introducing a specific DNA molecule into the producing strain (methanotrophic bacterium); the specific DNA molecule consists of an upstream homology arm, a resistance gene and a downstream homology arm; the upstream arm targets the upstream of the start codon of the coding gene of the test protein in the starting bacterium genome DNA, and the downstream arm targets the downstream of the stop codon of the coding gene of the test protein in the starting bacterium genome DNA.
The upstream homology arm may be 800-1200bp, and specifically may be 1000 bp.
The downstream homology arm can be 800-1200bp, and specifically can be 1000 bp.
The resistance gene may specifically be a kanamycin resistance gene.
Specifically, the kanamycin resistance gene is shown as the 1001-1816 th nucleotide in the sequence 5 of the sequence table.
The introduction of specific DNA molecules into the developing bacteria (methanotrophic bacteria) is achieved in particular by means of electroporation.
Electroporation was carried out using an electroporator.
Specifically, the electroporator conditions were set to: 1.5kV, 25. mu.F and 200. omega.
The specific DNA molecule introduction in the starting bacterium specifically comprises the following steps:
(1) culturing spawn running in 50mL liquid NMS culture medium containing 1% methanol to logarithmic phase; then, centrifuging and discarding the supernatant; then, resuspending the bacterial pellet with water, and centrifuging to remove the supernatant; then, resuspending the bacterial pellet with water to obtain a cell suspension;
(2) taking the cell suspension, adding specific DNA molecules and mixing; then, the mixture was transferred into an electric beaker and subjected to electroporation using an electroporator; then, resuscitating and culturing for 12-24 hours by adopting a liquid NMS culture medium containing 0.1% methanol; then, the supernatant was discarded by centrifugation, and the cells were plated on a solid NMS medium plate containing kanamycin and cultured at 30 ℃ for 4 to 7 days.
The specific DNA molecule introduction in the starting bacterium specifically comprises the following steps:
(1) 50mL of liquid NMS culture medium containing 1% (volume percentage content) methanol is adopted to culture spawn running till logarithmic growth phase (OD)600nmA value of 2); then, the mixture was centrifuged at 5000 Xg at 4 ℃ to obtain a solutionDiscarding the supernatant after 10 min; then, resuspending the bacterial cell sediment with 50mL of water, centrifuging for 10min at 4 ℃ and 5000 Xg, and discarding the supernatant; then, resuspending the bacterial cell sediment with 1mL of distilled water to obtain cell suspension;
(2) taking 50 mu L of cell suspension obtained in the step (1), adding 500ng of specific DNA molecules and gently mixing; then, the mixture was transferred into a low-temperature cuvette with a gap of 1 mm, and subjected to electroporation using an electroporator (conditions were set to 1.5kV, 25. mu.F, and 200. omega.); then, using 10mL liquid NMS culture medium containing 0.1% (volume percentage content) methanol to recover and culture for 12-24 hours at 30 ℃; then, the cells were centrifuged at 5000 Xg for 10min at room temperature, the supernatant was discarded, and the cell pellet was applied to a medium containing 50. mu.g/mL-1Kanamycin in solid NMS medium plate, 30 degrees C were cultured for 4-7 days.
The invention also protects the application of the NifA protein or the coding gene of the NifL protein or the NifL protein in regulating and controlling the growth of the methanotrophic bacteria by using nitrogen.
The modulation is positive modulation.
The abundance of NifA protein is reduced and the ability of methanotrophic bacteria to utilize nitrogen for bacterial growth is inhibited.
The NifA protein activity is reduced and the ability of methanotrophic bacteria to utilize nitrogen for bacterial growth is inhibited.
The coding gene of the NifA protein is knocked out, and methanotrophic bacteria lose the capacity of bacterial growth by using nitrogen.
The NifL protein abundance is reduced and the ability of methanotrophic bacteria to utilize nitrogen for bacterial growth is inhibited.
The NifL protein activity is reduced and the ability of methanotrophic bacteria to utilize nitrogen for bacterial growth is inhibited.
The coding gene of the NifL protein is knocked out, and the methanotrophic bacterium part loses the capacity of bacterial growth by using nitrogen.
The invention also protects the application of the NifA protein or the coding gene of the NifL protein or the NifL protein in regulating and controlling the methanotrophic bacteria by using nitrogen as a nitrogen source.
The modulation is positive modulation.
The abundance of NifA protein is reduced and the ability of methanotrophic bacteria to utilize nitrogen as a nitrogen source is inhibited.
The activity of NifA protein is reduced and the ability of methanotrophic bacteria to utilize nitrogen as a nitrogen source is inhibited.
The coding gene of the NifA protein is knocked out, and the methanotrophic bacterium loses the capability of utilizing nitrogen as a nitrogen source.
The NifL protein abundance was reduced and the methanotrophic bacteria ability to utilize nitrogen as a nitrogen source was inhibited.
The NifL protein activity was reduced and the methanotrophic bacteria ability to utilize nitrogen as a nitrogen source was inhibited.
The coding gene of the NifL protein was knocked out, and the methanotrophic bacterium portion lost the ability to utilize nitrogen as a nitrogen source.
The invention also protects the application of the NifA protein or the coding gene of the NifL protein or the NifL protein in regulating and controlling methanotrophic bacteria to carry out nitrogen fixation.
The modulation is positive modulation.
The abundance of the NifA protein is reduced, and the capability of methanotrophic bacteria for nitrogen fixation is inhibited.
The activity of the NifA protein is reduced, and the capability of methanotrophic bacteria for nitrogen fixation is inhibited.
The coding gene of the NifA protein is knocked out, and methanotrophic bacteria lose the capability of nitrogen fixation.
The NifL protein abundance was reduced and the methanotrophic bacteria ability to utilize nitrogen as a nitrogen source was inhibited.
The NifL protein activity was reduced and the methanotrophic bacteria ability to utilize nitrogen as a nitrogen source was inhibited.
The coding gene of the NifL protein was knocked out, and the methanotrophic bacterium portion lost the ability to utilize nitrogen as a nitrogen source.
The invention also protects the application of the NifA protein or the coding gene of the NifL protein or the NifL protein in participating in the bacterial growth of the methanotrophic bacteria by using nitrogen.
The NifA protein is (a1) or (a2) as follows:
(a1) protein shown in a sequence 1 in a sequence table;
(a2) a protein derived from methanotrophic bacterium, having 98% or more identity to (a1), and involved in the growth of methanotrophic bacterium by nitrogen gas;
the coding gene of NifA is (b1) or (b2) or (b3) or (b4) as follows:
(b1) the coding region is shown as the DNA molecule at the 1001 st and 2527 th nucleotides in the sequence 2 of the sequence table;
(b2) a DNA molecule shown in a sequence 2 of a sequence table;
(b3) a DNA molecule that hybridizes under stringent conditions to (b1) or (b2) and encodes the protein.
NifL proteins are (a1) or (a2) as follows:
(a1) protein shown in a sequence 3 in a sequence table;
(a2) a protein derived from methanotrophic bacterium, having 98% or more identity to (a1), and involved in the growth of methanotrophic bacterium by nitrogen gas;
the coding gene of NifL protein is (b1) or (b2) or (b3) or (b 4):
(b1) the coding region is a DNA molecule shown as 1001 st and 2590 th nucleotides in a sequence 4 of a sequence table;
(b2) a DNA molecule shown in a sequence 4 of a sequence table;
(b3) a DNA molecule that hybridizes under stringent conditions to (b1) or (b2) and encodes the protein.
The stringent conditions are hybridization and washing of the membrane 2 times 5min at 68 ℃ in a solution of 2 XSSC, 0.1% SDS and 2 times 15min at 68 ℃ in a solution of 0.5 XSSC, 0.1% SDS.
The specific DNA molecule can be specifically shown as a sequence 5 or a sequence 6 in a sequence table.
Any of the methanotrophic bacteria described above may be a bacterium of the class gamma-Proteobacteria (Gamma).
Any of the methanotrophic bacteria described above may be of the order Methylococcales (Methylococcales).
Any of the methanotrophic bacteria may be a bacterium of the methylcoccaceae family (Methylococcaceae).
Any of the methanotrophic bacteria may be a Methylomicbium (Methylomicbium) bacterium.
Any of the methanotrophic bacteria described above may be of the Methylomicbium buryatense species.
Any one of the methanotrophic bacteria may be m.
Compared with the application level of the traditional gene knockout technology, the invention utilizes the gene knockout technology which is improved and reconstructed to cause gene silencing to regulate and control the nitrogen fixation capability of the strain, and has novel mode, simple, convenient and fast method, high operation genetic stability and strong controllability.
Drawings
FIG. 1 is a graph showing the growth in example 1.
FIG. 2 is a graph showing the growth in example 2.
FIG. 3 is a graph showing the growth in example 3.
Detailed Description
The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.
The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified. Unless otherwise stated, the quantitative tests in the following examples were repeated in three batches, each of which was set to 2 replicates, and the results were averaged.
Liquid NMS culture medium: MgSO (MgSO)4·7H2O 1g，CaCl2·6H2O 0.02g，KNO31g, 7.5g NaCl, 20mL phosphate buffer solution, 50mL carbonate buffer solution and 1mL trace element solution, and distilled water is used for supplementing 1L. Phosphate buffer solution (ph 6.8): KH (Perkin Elmer)2PO4 5.44g·L-1And Na2HPO4 10.73g·L-1And the balance being water. Carbonate buffer solution: 1M NaHCO3700mL of aqueous solution and 1M Na2CO3300mL of the aqueous solution. Solution of trace elements: na (Na)2-EDTA·2H2O 1g·L-1，FeSO4·7H2O 2g·L-1，ZnSO4·7H2O 0.8g·L-1，MnCl2·4H2O0.03g·L-1，H3BO3 0.03g·L-1，CoCl2·6H2O 0.2g·L-1，CuCl2·2H2O 0.6g·L-1，NiCl2·6H2O 0.02g·L-1，Na2MO4·2H2O 0.05g·L-1And the balance of distilled water.
Solid NMS medium: the only difference from liquid NMS medium was the addition of 15g agar powder per liter.
Liquid hnss medium: KNO3The amount was changed to 0.5g, and the rest was NMS medium.
Example 1 discovery of growth characteristics of Methanophilus
The inventor finds that methanotrophic bacteria have the following properties: after the nitrogen source in the medium is exhausted, the bacterial load can be further increased by using nitrogen in the environment.
Test bacteria: m. buryatense strain 5GB 1.
Test groups: the sterilized injection bottle (500mL capacity) is filled with 100mL of sterilized liquid hNMS culture medium, and then the test bacteria is inoculated, the initial OD of the system600nmA value of 0.5; then vacuumizing the injection bottle, introducing a mixed gas of nitrogen and oxygen (the volume percentage of the oxygen in the mixed gas is 17.85%) to normal air pressure, and then sealing; then, the cells were cultured at 30 ℃ for 108 hours with shaking at 200 rpm. During the culture process, the following steps are carried out again every 12 hours: vacuumizing the injection bottle, introducing a mixed gas of nitrogen and oxygen (the volume percentage of the oxygen in the mixed gas is 17.85%) to normal pressure, and sealing.
Control group: helium was used instead of nitrogen, and the test groups were otherwise identical.
Detecting OD of liquid phase culture system every 12 hours in the culture process600nmValues, growth curves were plotted.
During the culture process, samples are taken every 12 hours, supernatant (namely culture supernatant) is collected by centrifugation, and the culture supernatant or diluent thereof (dilution solvent is RO water) is used as sample liquid to be detected to detect the concentration of nitrate ions (ultraviolet spectrophotometry). The specific method comprises the following steps: 0.5mL of sample solution to be tested (equal volume of RO water as blank control), 0.4mL of 1 mol.L-1Preparing 10mL of detection system from 0.1mL of 0.8% sulfamic acid aqueous solution (eliminating influence of nitrite) and 9mL of RO water, and measuring absorbance A at 275nm (eliminating absorption interference of organic substances) and 220nm (linear relationship between absorbance and concentration at the wavelength of nitrate) positions of an ultraviolet spectrophotometer by using a quartz constant cuvette with a 10mm optical pathSchool＝A220-2A275Absorbance ASchoolIs proportional to the nitrate content. Preparing potassium nitrate aqueous solution with gradient concentration, wherein the concentration of N element is respectively 40, 28, 20, 12 or 4 mg.L-1And measuring the absorbance according to the same operation steps of the sample measurement to obtain a standard curve. According to the standard curve, the concentration of nitrate ions in the culture supernatant was calculated.
The control group was cultured for 36 hours, and the nitrate ion concentration in the culture supernatant was 17.9 mg.L-1Then, the concentration of nitrate ions in the culture supernatant was 15-30 mg.L for 108 hours after the culture-1Within the range. The test group was cultured for 36 hours, and the nitrate ion concentration in the culture supernatant was 19.3 mg.L-1Then, the concentration of nitrate ions in the culture supernatant was 15-30 mg.L for 108 hours after the culture-1Within the range.
The growth curve is shown in FIG. 1. OD of control group after 48 hours of culture600nmThe value reaches the peak (OD)600nmValue 4.45), there was no significant increase thereafter, i.e. there was no significant increase in the amount of bacteria in the control group from 48 hours to 108 hours of culture. OD of test group after 48 hours of culture600nmThe value continued to rise until the peak (OD) was reached after 96 hours of culture600nmValue 6.22), i.e. fromThe bacterial load continued to increase from 48 hours to 96 hours of culture. The results show that: in the control group, along with the fact that the content of nitrate ions in a liquid phase culture system reaches a low value, the bacteria to be tested lack a nitrogen source, so that the increase of the bacterial quantity cannot be realized; in the test group, after the content of nitrate ions in the liquid phase culture system reaches a low value, the test bacteria can continuously realize the increase of the bacterial quantity by using nitrogen in the environment as a nitrogen source.
Example 2 functional verification of nifA Gene
Host bacteria: m. buryatense strain 5GB 1.
The nifA gene is a gene in NCBI, and the function of the Protein is unknown (Derived by automated comparative analysis using gene prediction method: Protein Homology). The sequence of the nifA gene coding region and partial nucleotides at the upstream and downstream of the nifA gene coding region in the host bacterium genome DNA are shown as a sequence 2 in the sequence table (the 1001- & ltth & gt and the 2527 th nucleotides are coding regions). NifA protein shown in sequence 1 of the nifA gene coding sequence table.
Based on nifA gene, a specific DNA molecule A is designed. The specific DNA molecule A is a double-stranded DNA molecule and is shown as a sequence 5 in a sequence table. In the sequence 5, the 1 st to 1000 th nucleotides are upstream homology arms (Al, consisting of 1000 nucleotides upstream of the initiation codon of nifA gene), the 1001 st and 1816 th nucleotides are kanamycin resistance genes (KanR gene), and the 1817 st and 2816 th nucleotides are downstream homology arms (Ar, consisting of 1000 nucleotides downstream of the termination codon of nifA gene). The specific DNA molecule A can be artificially synthesized or prepared by overlapping PCR.
1. 50mL of liquid NMS culture medium containing 1% (volume percentage) methanol is adopted to culture host bacteria cells to logarithmic growth phase (OD)600nmA value of 2); then, centrifuging at 4 ℃ and 5000 Xg for 10min, and discarding the supernatant; then, 50mL of cold water is used for resuspending the bacterial cell sediment, and then the bacterial cell sediment is centrifuged for 10min at the temperature of 4 ℃ and at the speed of 5000 Xg, and the supernatant is discarded; then, the bacterial cell pellet was resuspended in 1mL of distilled water to obtain a cell suspension.
2. Taking 50 mu L of the cell suspension obtained in the step 1, adding 500ng of the specific DNA molecule A and gently mixing; then, the mixture was transferred into a low-temperature cuvette with a gap of 1 mm, and electroporation was carried out using an electroporator (conditions were set)1.5kV, 25 muF and 200 omega); then, using 10mL liquid NMS culture medium containing 0.1% (volume percentage content) methanol to recover and culture for 12-24 hours at 30 ℃; then, the cells were centrifuged at 5000 Xg for 10min at room temperature, the supernatant was discarded, and the cell pellet was applied to a medium containing 50. mu.g/mL-1Kanamycin in solid NMS medium plate, 30 degrees C were cultured for 4-7 days.
3. After step 2 was completed, single colonies were picked and PCR-characterized using a primer pair consisting of p-nifA-F and p-nifA-R. The amplification product of the host bacterium is 1583 bp. If an amplified product of 872bp is obtained, the bacterium is a target recombinant bacterium.
p-nifA-F：5'-CGAAACGCTAAAACCAAACGATAAGCC-3'；
p-nifA-R：5'-AGGGATTTTGCTTGTTTCGTTAACCGAAT-3'。
4. Drawing growth curve
Test bacteria: a host bacterium (M.buryatense 5GB1) or a recombinant bacterium obtained in step 3 (M.buryatense 5GB 1. delta. nifA).
The sterilized injection bottle (500mL capacity) is filled with 100mL of sterilized liquid hNMS culture medium, and then the test bacteria is inoculated, the initial OD of the system600nmA value of 0.5; then vacuumizing the injection bottle, introducing a mixed gas of nitrogen and oxygen (the volume percentage of the oxygen in the mixed gas is 17.85%) to normal air pressure, and then sealing; then, the cells were cultured at 30 ℃ for 108 hours with shaking at 200 rpm. During the culture process, the following steps are carried out again every 12 hours: vacuumizing the injection bottle, introducing a mixed gas of nitrogen and oxygen (the volume percentage of the oxygen in the mixed gas is 17.85%) to normal pressure, and sealing.
Detecting OD of liquid phase culture system every 12 hours in the culture process600nmValues, growth curves were plotted.
The growth curve is shown in FIG. 2. OD of host bacteria after 48 hours of culture600nmThe value continued to rise until 84 hours of incubation reached the peak (OD)600nmThe value is 5.56), namely the bacterial quantity continuously increases from 48 hours to 84 hours of culture, namely the host bacteria can carry out nitrogen metabolism after the nitrogen source in the culture medium is exhausted. Culturing for 48 hours, and recombining OD of bacteria600nmThe value reaches the peak (OD)600nmA value of 3.63) and then rapidly decreases (enters a decay phase), i.e. the recombinant bacteria cannot carry out nitrogen metabolism after the nitrogen source in the culture medium is exhausted and thus enters the decay phase. The results show that: the protein coded by the nifA gene participates in the growth of bacterial quantity by using nitrogen in the environment as a nitrogen source, and the bacterial strain loses the capability of using the nitrogen in the environment as the nitrogen source (namely loses the nitrogen fixation capability) after the nifA gene is knocked out.
Example 3 functional verification of nifL Gene
Host bacteria: m. buryatense strain 5GB 1.
The nifL gene is a gene in NCBI, and the function of the Protein is unknown (Derived by automated comparative analysis using gene prediction method: Protein Homology). Through sequencing verification, the nifL gene coding region and partial nucleotides at the upstream and downstream of the nifL gene coding region in the host bacterium genome DNA are shown as a sequence 4 in a sequence table (the 1001- & ltSUB & gt 2590 & ltSUB & gt nucleotide is a coding region). nifL protein shown in a sequence 3 of a nifL gene coding sequence table.
Based on nifL gene, a specific DNA molecule B is designed. The specific DNA molecule B is a double-stranded DNA molecule and is shown as a sequence 6 in a sequence table. In the sequence 6, the 1 st to 1000 th nucleotides are upstream homology arms (Ll, consisting of 1000 nucleotides upstream of the initiation codon of nifL gene), the 1001 st and 1816 th nucleotides are kanamycin resistance genes (KanR gene), and the 1817 st and 2816 th nucleotides are downstream homology arms (Lr, consisting of 1000 nucleotides downstream of the termination codon of nifL gene). The specific DNA molecule B can be artificially synthesized or prepared by overlapping PCR.
1. Same as in step 1 of example 2.
2. The procedure was as in step 2 of example 2 except that the specific DNA molecule A was replaced with the specific DNA molecule B.
3. After step 2 was completed, a single colony was picked and PCR-identified using a primer pair consisting of p-nifL-F and p-nifL-R. The amplification product of the host bacterium is 1634 bp. If 860bp of amplification product is obtained, the bacterium is a target recombinant bacterium.
p-nifL-F：5'-ACCCATCCCAACGATATCGAGCAA-3’；
p-nifL-R：5'-AGCAGACGTTCGGACATGGC-3’。
4. Drawing growth curve
Test bacteria: a host bacterium (M.buryatense 5GB1) or a recombinant bacterium obtained in step 3 (M.buryatense 5GB 1. delta. nifL).
The procedure is as in step 4 of example 2.
The growth curve is shown in FIG. 3. OD of host bacteria after 48 hours of culture600nmThe value continued to rise until 84 hours of incubation reached the peak (OD)600nmThe value is 5.56), namely the bacterial quantity continuously increases from 48 hours to 84 hours of culture, namely the host bacteria can carry out nitrogen metabolism after the nitrogen source in the culture medium is exhausted. After 72 hours of culture, the OD of the recombinant bacteria600nmThe value reaches the peak (OD)600nmA value of 4.40), then rapidly decreases (enters a decay phase), namely the recombinant bacteria can carry out a certain degree of nitrogen metabolism after the nitrogen source in the culture medium is exhausted, but the growth condition of the recombinant bacteria is inferior to that of the host bacteria. The results show that: the protein encoded by the nifL gene participates in the bacterial strain to realize the increase of bacterial load by using nitrogen in the environment as a nitrogen source, and the bacterial strain partially loses the capability of using the nitrogen in the environment as the nitrogen source (namely partially loses the nitrogen fixing capability) after the nifL gene is knocked out.
The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.
Sequence listing
<110> university of west ampere traffic
<120> method for modifying and regulating nitrogen-fixing microorganism
<130> GNCYX211483
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 508
<212> PRT
<213> Methylomicrobium buryatense
<400> 1
Met Ser Glu Arg Leu Leu Leu Val Glu Ser Glu Leu Asp Thr Leu Phe
1 5 10 15
Leu Val Ser Gln Leu Leu Asn Ser Thr Arg Asp Leu Arg Asp Lys Leu
20 25 30
Arg Gly Ile Leu Glu Ile Leu His Lys Arg Asn Gly Leu His Phe Gly
35 40 45
Met Ile Thr Leu Arg Glu Val Asp Asp Asp Ser Met Ser Ile Cys Glu
50 55 60
Val Tyr Gly Asp Asn Ile Asp Lys Ser Val Arg Tyr Gln Pro Gly Glu
65 70 75 80
Gly Leu Val Gly Ala Ile Leu Asp Glu Gly Ser Thr Ile Val Val Asp
85 90 95
Arg Ile Thr Asp Glu Pro Arg Phe Leu Ser Arg Leu Gly Val Tyr Asn
100 105 110
Arg Asp Leu Pro Phe Ile Gly Ser Pro Leu Ala Val Asp Gln Gly Glu
115 120 125
Val Val Gly Ile Leu Ala Ala Gln Pro Ala Glu Ala Ser Phe Leu Gly
130 135 140
Glu Lys Ala Arg Phe Leu Glu Met Val Ala Asn Leu Ile Ala Gln Ser
145 150 155 160
Val Asn Ser Leu Arg Ala Ile Glu Arg Lys Gln Asp Gln Leu Thr Asn
165 170 175
Glu Arg Asp Tyr Leu Lys Gln Glu Leu Val Lys Asn Tyr Arg Phe Glu
180 185 190
Asn Ile Ile Gly His Ser Glu Pro Met Leu Lys Val Phe Asp Ile Ile
195 200 205
Arg Gln Val Ala Lys Trp Asn Thr Thr Val Leu Ile Arg Gly Glu Ser
210 215 220
Gly Thr Gly Lys Glu Val Val Ala Asn Ala Ile His Phe Asn Ser Ala
225 230 235 240
Cys Ala Asn Gly Pro Phe Leu Lys Leu Asn Cys Ala Ala Leu Pro Asp
245 250 255
Thr Leu Leu Glu Ser Glu Leu Phe Gly His Glu Lys Gly Ala Phe Ser
260 265 270
Gly Ala Ile Gly Gln Arg Lys Gly Arg Phe Glu Leu Ala Asp Asn Gly
275 280 285
Thr Leu Phe Leu Asp Glu Ile Gly Glu Ile Ser Ala Ser Phe Gln Ala
290 295 300
Lys Leu Leu Arg Val Leu Gln Glu Gly Glu Phe Glu Arg Val Gly Gly
305 310 315 320
Val Lys Thr Leu Lys Val His Val Arg Val Ile Ala Ala Thr Asn Arg
325 330 335
Asn Leu Glu Glu Glu Val Ala Glu Gly Arg Phe Arg Glu Asp Leu Tyr
340 345 350
Tyr Arg Leu Asn Val Met Pro Ile Asn Met Pro Ala Leu Arg Glu Arg
355 360 365
Ile Glu Asp Ile Pro Glu Leu Ala Gln Phe Leu Leu Lys Arg Ile Ser
370 375 380
Lys Gln Gln Ala Gly Arg Pro Leu Glu Ile Lys Glu Ser Ala Ile Arg
385 390 395 400
Ile Leu Met Lys His His Trp Pro Gly Asn Val Arg Glu Leu Glu Asn
405 410 415
Arg Leu Glu Arg Ala Ala Ile Met Ser Gln Asp Gly Ile Ile Asp Arg
420 425 430
Asp Val Ile Ala Gly Thr Gly Leu Glu Ser Glu Ile Gly Ile Thr Leu
435 440 445
Ala Pro Gln Pro Val Arg Ser Met Asp Leu Leu Asp Asp Asn Leu Asp
450 455 460
Glu Arg Glu Arg Val Ile Ala Ala Leu Glu Gln Ser Gly Trp Val Gln
465 470 475 480
Ala Lys Ala Ala Arg Leu Leu Asp Met Thr Pro Arg Gln Ile Ala Tyr
485 490 495
Arg Ile Gln Thr Leu Lys Ile His Val Lys Gln Ile
500 505
<210> 2
<211> 3527
<212> DNA
<213> Methylomicrobium buryatense
<400> 2
accagtgttg ttttttttga agttattacg ccaggacatg ggcgatctat ggcaatacgc 60
ggcccgaaaa gaacgcggtt tcgtcaacag agaacttcga atcgaaggca tcgataagcg 120
cgggacgcgg tggtacgcat gctccggtaa ctggttcgcc gaaaaggaaa ccggcgccga 180
cgcatttttt tccggggaaa ccaaagatta tttactgctc agtttcagcg atatcacgca 240
acaacgccgc cagcaggagg aattgcacct acagaatctt cgcaccattt tggccgagga 300
agagcaaatc cgtagcatcc gggaaacctt actcggcgcg atgcaccaaa ttaggcaacc 360
gttaaatcaa attagtgcag caatcaacat catgagtcat cgcaacgata ttcaaaacca 420
accgttgaga gaactgctcg aacaagtcgg tacgaaaggc gaagagactc tggaaacctt 480
acagcgatgc gttcccgaaa tcccggaaac cgccgtgatt ccggtcaatt tgaaccaaat 540
tctgcatgag gtcgtggagc taagtagtta taagcttctg gccaacggga tcgtcatcga 600
ttggcggccc agttcgacgc tgccttcggt attgggttcg gaaaacaagc tgcgcatgct 660
attcaaacag ttaatcgata acgcgataga agcaatgaac cgcgcaggga gccgcgaacg 720
ttccattgct attgcaacgc atgtcgacaa cgatcgagtc aatgtgtcga ttgaagatac 780
cggcccaggt atccatccgg accagcgcat caaggtattt caaccttttt acacgacccg 840
cccgatgggc acgatcaagg caggcatggg tttagtcatg gtccaggaaa tcgtcaatca 900
acataaagcc ggcatcgaaa tcgatccgaa ttacgatcaa ggttgccgat ttaaaatcgg 960
ttttccaatc tatcgaaacg ctaaaaccaa acgataagcc atgtccgaac gtctgctatt 1020
agtcgaatcc gaattggata cgctgtttct agtcagccaa ctattgaaca gcacccgaga 1080
tttacgcgat aaattgcggg gtatccttga aatattacat aaacgcaacg gccttcattt 1140
cggcatgatc acattgcgtg aggtcgatga cgacagcatg agcatctgcg aagtctatgg 1200
cgacaacatc gataagtcgg tgcgttatca gcccggggaa ggactggtcg gtgcgatact 1260
ggacgaaggc agcacgatcg tggtcgacag aatcaccgac gaaccgcgtt tcttgagccg 1320
cctgggcgtc tataatcgcg acttgccttt catcggctct ccgttggccg tggaccaagg 1380
tgaggtcgta ggtattctag ccgcacaacc ggccgaagcg agttttctcg gtgagaaggc 1440
ccgctttctc gagatggtcg cgaacctgat cgcgcaaagc gtcaattcgc tcagggcgat 1500
cgagcgaaaa caagatcaat tgaccaacga acgcgattat ctaaaacagg aactagtcaa 1560
aaactaccgt ttcgaaaaca tcatcggcca ctccgagccg atgctgaagg tattcgacat 1620
catccggcaa gtcgcgaaat ggaacacgac cgtgctgata cgcggcgagt cgggcaccgg 1680
caaagaagtc gtcgcgaacg cgatccattt caattcagcc tgcgcgaatg gcccgttttt 1740
gaaattgaat tgcgcggccc tgcccgacac cttgctcgaa tccgaactgt tcggccacga 1800
gaaaggcgcg ttcagcggcg cgatcggtca gcgcaagggg cgtttcgaac tcgccgacaa 1860
cggcaccttg tttctcgacg aaatcggcga aatttcggcc tcgttccaag ccaaattatt 1920
acgggtgctg caagagggcg agttcgaacg tgtcggcggc gttaaaacct taaaagtgca 1980
tgtacgggta atcgccgcaa cgaaccgcaa tcttgaagaa gaggtcgccg aaggacggtt 2040
tcgcgaggac ttgtattacc ggctcaatgt catgccgatt aatatgccgg cgctacgcga 2100
gcgtatcgaa gatattcccg aattggcaca atttttactc aaacgtattt ccaaacaaca 2160
agccggacgg ccgctcgaaa tcaaagaaag tgcgattcgt atcttgatga agcatcattg 2220
gcccggcaat gtccgggaat tggaaaaccg gctcgaacgc gccgcgatca tgagtcagga 2280
cggcatcatc gaccgagatg tgatcgccgg caccggtttg gaaagcgaaa taggcattac 2340
gctagcaccg cagcctgtcc gtagcatgga tttactcgat gataacctag acgaacgcga 2400
acgcgtcatc gccgcactcg aacaaagcgg ctgggtacaa gccaaagcag cgcgcctgct 2460
cgacatgacg ccgagacaaa tcgcttaccg aattcaaaca ctcaaaattc atgtgaagca 2520
gatttgaatt cggttaacga aacaagcaaa atccctttta cgcaaccgat gccgaccatt 2580
gccgagcatt gccacagtac caatttaacc ggcctgtcga ccgaatgtat cgctagcgtc 2640
gtatttaacg aacaccccaa accgctgcat atcgccggaa cccgagaagc cgcttcgggc 2700
cttttcgaac tcttggcgaa acaatcggcg cccgagacat gcggccgttt gtttcaggac 2760
tatatgtgcg tcgtattcgg cttcgaatcc gagcagcgct tagcggacga cgtaacggga 2820
gggcggcgtt accgcaatag ctatttgcgc ttgatacagg attggggaat ggactcgaac 2880
aatgcacaag cggcggtttt caaaggttgg gtcgaaagcc gtttcggttt gtttccgtcc 2940
tatcacaagc aacccattac cggcttcggc acgaaggcct ggatcggcta tatcgaagaa 3000
aagatgaaca gccgatatca caataactgc atctatatgc aattggacct gctctacgaa 3060
tattgtcaat ggatcatcga acgattccgc tttccggctg caggacataa gacgctgtat 3120
cgaggggtcg atacgctgga cgactgcatc atccggcatg aaaccaaaca cgataaaatc 3180
gtgcgtttta acaatctggt atcgtttacc gataagccgg gcaccgccag cgaattcgga 3240
gcttatattc tgaagaccga agtaccgatg gtaaaattga tttttttcaa cgacctactg 3300
ccccgccacg ccttgcgtgg agaagcggaa tatttggcaa tcggcggcgc ttaccgggtc 3360
cgggtaagtc attgaggcga actaaggata cggtaaaaaa aataactccc ctaaatgttt 3420
cttacgcctt gtgcatatcc cgcacaagtt tatcgttccc atgctctgcg tcactgccat 3480
taagttacct gattagcaag tttcttcagc taaagcccaa aaaccag 3527
<210> 3
<211> 529
<212> PRT
<213> Methylomicrobium buryatense
<400> 3
Met Met Thr Leu Phe Ala Pro Ser Leu Thr Ile Asp Lys Gln Leu Leu
1 5 10 15
Asp Leu Ile Glu Ser Phe Ser Arg Gln Gly Lys Met Leu Pro Phe Phe
20 25 30
Leu Leu Ile Lys Val Val Glu Gln Val Pro Ile Ala Ile Ser Ile Thr
35 40 45
Asp Lys Lys Ala Asn Ile Leu Tyr Ile Asn Gln Ala Phe Thr Asp Thr
50 55 60
Thr Gly Tyr Arg Pro Ala Glu Ile Leu Gly Arg Asn Glu Ser Glu Leu
65 70 75 80
Ser Ala Lys Ser Thr Pro Arg Gln Val Tyr Tyr Asp Leu Trp His Thr
85 90 95
Ile Thr Arg Lys Lys Ile Trp Arg Gly Arg Leu Ile Asn Lys Asn Lys
100 105 110
Asn Gly Ala Pro Tyr Leu Ala Glu Leu Thr Ile Ala Pro Met Gln Asp
115 120 125
Glu Gln Gly Lys Ile Thr His Tyr Ile Gly Met His Arg Asp Ile Thr
130 135 140
Gln Thr His Ala Ser Glu Gln Lys Leu Ile Asn Gln Lys Gln Leu Ile
145 150 155 160
Glu Ser Val Ile Asn Ala Ser Pro Val Ala Met Val Val Ile Asp Lys
165 170 175
Asn Asp Arg Val Val Leu Asp Asn Gln Val Tyr Lys Met Met Ile Ser
180 185 190
Asp Leu Gly Leu Thr Glu Pro Val Leu Phe Phe Leu Lys Leu Leu Arg
195 200 205
Gln Asp Met Gly Asp Leu Trp Gln Tyr Ala Ala Arg Lys Glu Arg Gly
210 215 220
Phe Val Asn Arg Glu Leu Arg Ile Glu Gly Ile Asp Lys Arg Gly Thr
225 230 235 240
Arg Trp Tyr Ala Cys Ser Gly Asn Trp Phe Ala Glu Lys Glu Thr Gly
245 250 255
Ala Asp Ala Phe Phe Ser Gly Glu Thr Lys Asp Tyr Leu Leu Leu Ser
260 265 270
Phe Ser Asp Ile Thr Gln Gln Arg Arg Gln Gln Glu Glu Leu His Leu
275 280 285
Gln Asn Leu Arg Thr Ile Leu Ala Glu Glu Glu Gln Ile Arg Ser Ile
290 295 300
Arg Glu Thr Leu Leu Gly Ala Met His Gln Ile Arg Gln Pro Leu Asn
305 310 315 320
Gln Ile Ser Ala Ala Ile Asn Ile Met Ser His Arg Asn Asp Ile Gln
325 330 335
Asn Gln Pro Leu Arg Glu Leu Leu Glu Gln Val Gly Thr Lys Gly Glu
340 345 350
Glu Thr Leu Glu Thr Leu Gln Arg Cys Val Pro Glu Ile Pro Glu Thr
355 360 365
Ala Val Ile Pro Val Asn Leu Asn Gln Ile Leu His Glu Val Val Glu
370 375 380
Leu Ser Ser Tyr Lys Leu Leu Ala Asn Gly Ile Val Ile Asp Trp Arg
385 390 395 400
Pro Ser Ser Thr Leu Pro Ser Val Leu Gly Ser Glu Asn Lys Leu Arg
405 410 415
Met Leu Phe Lys Gln Leu Ile Asp Asn Ala Ile Glu Ala Met Asn Arg
420 425 430
Ala Gly Ser Arg Glu Arg Ser Ile Ala Ile Ala Thr His Val Asp Asn
435 440 445
Asp Arg Val Asn Val Ser Ile Glu Asp Thr Gly Pro Gly Ile His Pro
450 455 460
Asp Gln Arg Ile Lys Val Phe Gln Pro Phe Tyr Thr Thr Arg Pro Met
465 470 475 480
Gly Thr Ile Lys Ala Gly Met Gly Leu Val Met Val Gln Glu Ile Val
485 490 495
Asn Gln His Lys Ala Gly Ile Glu Ile Asp Pro Asn Tyr Asp Gln Gly
500 505 510
Cys Arg Phe Lys Ile Gly Phe Pro Ile Tyr Arg Asn Ala Lys Thr Lys
515 520 525
Arg
<210> 4
<211> 3590
<212> DNA
<213> Methylomicrobium buryatense
<400> 4
cttgctatcg cggataatac gtacataaag ccccctgtct aacttttaaa gtccggcaaa 60
acccataaag gcgagagaga gaattccggc ggttatgaat gcgatcggcg taccgtcaaa 120
taataccggt accgtcatta aagaaagacg ctctctaagt cctgcaaaaa taaccatcac 180
cagcgtaaac cccaatgccg acccgaaacc gaacatgacg ctctgcaaga aattcgaatc 240
gtgttgaata ttcagcaaag cgacgccaag caccgcacaa ttcgtggtaa tgaggggcaa 300
gaatattccc aatacttgat aaagcaccgg gctggtttta tgaatgacca tttcggtgaa 360
ctgtacaact gccgcaatca ccaagataaa acccagtacg cgcatgaagc cgatatcgag 420
cggtatcaat accccgtgtt ccaacaccca accggcagaa gccgccaacg tcaatacgaa 480
ggttgtcgcc aagcccatcc ccaacgccgt atccagctta ttggagactc ccataaacgg 540
gcacaatccc agaaacttga ccaacacgac gttgttgacc aatgccgtgc ctagaatcaa 600
catgaaataa tcgcccattg cccctaccct acaaattgcc tctaatatcg gcggagcagc 660
aaacgtgcca ataagtcgac caagcttgca aatcccggaa tatcaaggtt aagaataatg 720
gcggcatcgg accgactgtt ggaaattgaa cagtcgcctt caatcctcgc gcctcattgt 780
cgtaaaactt acaaatccaa gcatttttat ccggtaataa aaatgtccta cggcaagcat 840
ccccctcctt ccaattttca aatctggcga ttgttttcag cctaatcgaa attggcttct 900
aatttgcata ttccttttta ttattatgga aattacgaga ccgatcgata tgaacaatac 960
gcttcccggt tacgaaaccc atcccaacga tatcgagcaa atgatgacac tattcgctcc 1020
gtcgttgacg atcgataaac agctattgga tttgatcgaa tcgtttagcc gtcaaggcaa 1080
gatgctcccc ttttttctat tgatcaaagt tgtcgagcaa gtaccgatcg ccatatcgat 1140
caccgacaaa aaagccaaca ttctctacat taatcaagca tttaccgata ccaccggcta 1200
ccgtcccgca gaaatcctcg gacgtaatga atccgagtta tcggccaaat cgacaccccg 1260
ccaggtctat tacgatttgt ggcacacaat cacccgcaaa aaaatctggc gcggccgttt 1320
gatcaacaaa aataaaaacg gagccccata tcttgcagag ctcacgattg cgcccatgca 1380
ggacgaacag ggaaaaatca cgcattacat cggcatgcac cgggatatca cccaaacaca 1440
cgcctccgaa caaaagttaa tcaatcagaa gcaattgatc gaatcggtga tcaatgcatc 1500
gccggtagcg atggtggtca tcgataagaa cgatcgcgtc gtcctggata accaagtata 1560
taaaatgatg atcagcgatc tgggcttgac tgaaccagtg ttgttttttt tgaagttatt 1620
acgccaggac atgggcgatc tatggcaata cgcggcccga aaagaacgcg gtttcgtcaa 1680
cagagaactt cgaatcgaag gcatcgataa gcgcgggacg cggtggtacg catgctccgg 1740
taactggttc gccgaaaagg aaaccggcgc cgacgcattt ttttccgggg aaaccaaaga 1800
ttatttactg ctcagtttca gcgatatcac gcaacaacgc cgccagcagg aggaattgca 1860
cctacagaat cttcgcacca ttttggccga ggaagagcaa atccgtagca tccgggaaac 1920
cttactcggc gcgatgcacc aaattaggca accgttaaat caaattagtg cagcaatcaa 1980
catcatgagt catcgcaacg atattcaaaa ccaaccgttg agagaactgc tcgaacaagt 2040
cggtacgaaa ggcgaagaga ctctggaaac cttacagcga tgcgttcccg aaatcccgga 2100
aaccgccgtg attccggtca atttgaacca aattctgcat gaggtcgtgg agctaagtag 2160
ttataagctt ctggccaacg ggatcgtcat cgattggcgg cccagttcga cgctgccttc 2220
ggtattgggt tcggaaaaca agctgcgcat gctattcaaa cagttaatcg ataacgcgat 2280
agaagcaatg aaccgcgcag ggagccgcga acgttccatt gctattgcaa cgcatgtcga 2340
caacgatcga gtcaatgtgt cgattgaaga taccggccca ggtatccatc cggaccagcg 2400
catcaaggta tttcaacctt tttacacgac ccgcccgatg ggcacgatca aggcaggcat 2460
gggtttagtc atggtccagg aaatcgtcaa tcaacataaa gccggcatcg aaatcgatcc 2520
gaattacgat caaggttgcc gatttaaaat cggttttcca atctatcgaa acgctaaaac 2580
caaacgataa gccatgtccg aacgtctgct attagtcgaa tccgaattgg atacgctgtt 2640
tctagtcagc caactattga acagcacccg agatttacgc gataaattgc ggggtatcct 2700
tgaaatatta cataaacgca acggccttca tttcggcatg atcacattgc gtgaggtcga 2760
tgacgacagc atgagcatct gcgaagtcta tggcgacaac atcgataagt cggtgcgtta 2820
tcagcccggg gaaggactgg tcggtgcgat actggacgaa ggcagcacga tcgtggtcga 2880
cagaatcacc gacgaaccgc gtttcttgag ccgcctgggc gtctataatc gcgacttgcc 2940
tttcatcggc tctccgttgg ccgtggacca aggtgaggtc gtaggtattc tagccgcaca 3000
accggccgaa gcgagttttc tcggtgagaa ggcccgcttt ctcgagatgg tcgcgaacct 3060
gatcgcgcaa agcgtcaatt cgctcagggc gatcgagcga aaacaagatc aattgaccaa 3120
cgaacgcgat tatctaaaac aggaactagt caaaaactac cgtttcgaaa acatcatcgg 3180
ccactccgag ccgatgctga aggtattcga catcatccgg caagtcgcga aatggaacac 3240
gaccgtgctg atacgcggcg agtcgggcac cggcaaagaa gtcgtcgcga acgcgatcca 3300
tttcaattca gcctgcgcga atggcccgtt tttgaaattg aattgcgcgg ccctgcccga 3360
caccttgctc gaatccgaac tgttcggcca cgagaaaggc gcgttcagcg gcgcgatcgg 3420
tcagcgcaag gggcgtttcg aactcgccga caacggcacc ttgtttctcg acgaaatcgg 3480
cgaaatttcg gcctcgttcc aagccaaatt attacgggtg ctgcaagagg gcgagttcga 3540
acgtgtcggc ggcgttaaaa ccttaaaagt gcatgtacgg gtaatcgccg 3590
<210> 5
<211> 2816
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
accagtgttg ttttttttga agttattacg ccaggacatg ggcgatctat ggcaatacgc 60
ggcccgaaaa gaacgcggtt tcgtcaacag agaacttcga atcgaaggca tcgataagcg 120
cgggacgcgg tggtacgcat gctccggtaa ctggttcgcc gaaaaggaaa ccggcgccga 180
cgcatttttt tccggggaaa ccaaagatta tttactgctc agtttcagcg atatcacgca 240
acaacgccgc cagcaggagg aattgcacct acagaatctt cgcaccattt tggccgagga 300
agagcaaatc cgtagcatcc gggaaacctt actcggcgcg atgcaccaaa ttaggcaacc 360
gttaaatcaa attagtgcag caatcaacat catgagtcat cgcaacgata ttcaaaacca 420
accgttgaga gaactgctcg aacaagtcgg tacgaaaggc gaagagactc tggaaacctt 480
acagcgatgc gttcccgaaa tcccggaaac cgccgtgatt ccggtcaatt tgaaccaaat 540
tctgcatgag gtcgtggagc taagtagtta taagcttctg gccaacggga tcgtcatcga 600
ttggcggccc agttcgacgc tgccttcggt attgggttcg gaaaacaagc tgcgcatgct 660
attcaaacag ttaatcgata acgcgataga agcaatgaac cgcgcaggga gccgcgaacg 720
ttccattgct attgcaacgc atgtcgacaa cgatcgagtc aatgtgtcga ttgaagatac 780
cggcccaggt atccatccgg accagcgcat caaggtattt caaccttttt acacgacccg 840
cccgatgggc acgatcaagg caggcatggg tttagtcatg gtccaggaaa tcgtcaatca 900
acataaagcc ggcatcgaaa tcgatccgaa ttacgatcaa ggttgccgat ttaaaatcgg 960
ttttccaatc tatcgaaacg ctaaaaccaa acgataagcc atgagccata ttcaacggga 1020
aacgtcttgc tcgaggccgc gattaaattc caacatggat gctgatttat atgggtataa 1080
atgggctcgc gataatgtcg ggcaatcagg tgcgacaatc tatcgattgt atgggaagcc 1140
cgatgcgcca gagttgtttc tgaaacatgg caaaggtagc gttgccaatg atgttacaga 1200
tgagatggtc agactaaact ggctgacgga atttatgcct cttccgacca tcaagcattt 1260
tatccgtact cctgatgatg catggttact caccactgcg atccccggga aaacagcatt 1320
ccaggtatta gaagaatatc ctgattcagg tgaaaatatt gttgatgcgc tggcagtgtt 1380
cctgcgccgg ttgcattcga ttcctgtttg taattgtcct tttaacagcg atcgcgtatt 1440
tcgtctcgct caggcgcaat cacgaatgaa taacggtttg gttgatgcga gtgattttga 1500
tgacgagcgt aatggctggc ctgttgaaca agtctggaaa gaaatgcata agcttttgcc 1560
attctcaccg gattcagtcg tcactcatgg tgatttctca cttgataacc ttatttttga 1620
cgaggggaaa ttaataggtt gtattgatgt tggacgagtc ggaatcgcag accgatacca 1680
ggatcttgcc atcctatgga actgcctcgg tgagttttct ccttcattac agaaacggct 1740
ttttcaaaaa tatggtattg ataatcctga tatgaataaa ttgcagtttc atttgatgct 1800
cgatgagttt ttctaaattc ggttaacgaa acaagcaaaa tcccttttac gcaaccgatg 1860
ccgaccattg ccgagcattg ccacagtacc aatttaaccg gcctgtcgac cgaatgtatc 1920
gctagcgtcg tatttaacga acaccccaaa ccgctgcata tcgccggaac ccgagaagcc 1980
gcttcgggcc ttttcgaact cttggcgaaa caatcggcgc ccgagacatg cggccgtttg 2040
tttcaggact atatgtgcgt cgtattcggc ttcgaatccg agcagcgctt agcggacgac 2100
gtaacgggag ggcggcgtta ccgcaatagc tatttgcgct tgatacagga ttggggaatg 2160
gactcgaaca atgcacaagc ggcggttttc aaaggttggg tcgaaagccg tttcggtttg 2220
tttccgtcct atcacaagca acccattacc ggcttcggca cgaaggcctg gatcggctat 2280
atcgaagaaa agatgaacag ccgatatcac aataactgca tctatatgca attggacctg 2340
ctctacgaat attgtcaatg gatcatcgaa cgattccgct ttccggctgc aggacataag 2400
acgctgtatc gaggggtcga tacgctggac gactgcatca tccggcatga aaccaaacac 2460
gataaaatcg tgcgttttaa caatctggta tcgtttaccg ataagccggg caccgccagc 2520
gaattcggag cttatattct gaagaccgaa gtaccgatgg taaaattgat ttttttcaac 2580
gacctactgc cccgccacgc cttgcgtgga gaagcggaat atttggcaat cggcggcgct 2640
taccgggtcc gggtaagtca ttgaggcgaa ctaaggatac ggtaaaaaaa ataactcccc 2700
taaatgtttc ttacgccttg tgcatatccc gcacaagttt atcgttccca tgctctgcgt 2760
cactgccatt aagttacctg attagcaagt ttcttcagct aaagcccaaa aaccag 2816
<210> 6
<211> 2816
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
cttgctatcg cggataatac gtacataaag ccccctgtct aacttttaaa gtccggcaaa 60
acccataaag gcgagagaga gaattccggc ggttatgaat gcgatcggcg taccgtcaaa 120
taataccggt accgtcatta aagaaagacg ctctctaagt cctgcaaaaa taaccatcac 180
cagcgtaaac cccaatgccg acccgaaacc gaacatgacg ctctgcaaga aattcgaatc 240
gtgttgaata ttcagcaaag cgacgccaag caccgcacaa ttcgtggtaa tgaggggcaa 300
gaatattccc aatacttgat aaagcaccgg gctggtttta tgaatgacca tttcggtgaa 360
ctgtacaact gccgcaatca ccaagataaa acccagtacg cgcatgaagc cgatatcgag 420
cggtatcaat accccgtgtt ccaacaccca accggcagaa gccgccaacg tcaatacgaa 480
ggttgtcgcc aagcccatcc ccaacgccgt atccagctta ttggagactc ccataaacgg 540
gcacaatccc agaaacttga ccaacacgac gttgttgacc aatgccgtgc ctagaatcaa 600
catgaaataa tcgcccattg cccctaccct acaaattgcc tctaatatcg gcggagcagc 660
aaacgtgcca ataagtcgac caagcttgca aatcccggaa tatcaaggtt aagaataatg 720
gcggcatcgg accgactgtt ggaaattgaa cagtcgcctt caatcctcgc gcctcattgt 780
cgtaaaactt acaaatccaa gcatttttat ccggtaataa aaatgtccta cggcaagcat 840
ccccctcctt ccaattttca aatctggcga ttgttttcag cctaatcgaa attggcttct 900
aatttgcata ttccttttta ttattatgga aattacgaga ccgatcgata tgaacaatac 960
gcttcccggt tacgaaaccc atcccaacga tatcgagcaa atgagccata ttcaacggga 1020
aacgtcttgc tcgaggccgc gattaaattc caacatggat gctgatttat atgggtataa 1080
atgggctcgc gataatgtcg ggcaatcagg tgcgacaatc tatcgattgt atgggaagcc 1140
cgatgcgcca gagttgtttc tgaaacatgg caaaggtagc gttgccaatg atgttacaga 1200
tgagatggtc agactaaact ggctgacgga atttatgcct cttccgacca tcaagcattt 1260
tatccgtact cctgatgatg catggttact caccactgcg atccccggga aaacagcatt 1320
ccaggtatta gaagaatatc ctgattcagg tgaaaatatt gttgatgcgc tggcagtgtt 1380
cctgcgccgg ttgcattcga ttcctgtttg taattgtcct tttaacagcg atcgcgtatt 1440
tcgtctcgct caggcgcaat cacgaatgaa taacggtttg gttgatgcga gtgattttga 1500
tgacgagcgt aatggctggc ctgttgaaca agtctggaaa gaaatgcata agcttttgcc 1560
attctcaccg gattcagtcg tcactcatgg tgatttctca cttgataacc ttatttttga 1620
cgaggggaaa ttaataggtt gtattgatgt tggacgagtc ggaatcgcag accgatacca 1680
ggatcttgcc atcctatgga actgcctcgg tgagttttct ccttcattac agaaacggct 1740
ttttcaaaaa tatggtattg ataatcctga tatgaataaa ttgcagtttc atttgatgct 1800
cgatgagttt ttctaagcca tgtccgaacg tctgctatta gtcgaatccg aattggatac 1860
gctgtttcta gtcagccaac tattgaacag cacccgagat ttacgcgata aattgcgggg 1920
tatccttgaa atattacata aacgcaacgg ccttcatttc ggcatgatca cattgcgtga 1980
ggtcgatgac gacagcatga gcatctgcga agtctatggc gacaacatcg ataagtcggt 2040
gcgttatcag cccggggaag gactggtcgg tgcgatactg gacgaaggca gcacgatcgt 2100
ggtcgacaga atcaccgacg aaccgcgttt cttgagccgc ctgggcgtct ataatcgcga 2160
cttgcctttc atcggctctc cgttggccgt ggaccaaggt gaggtcgtag gtattctagc 2220
cgcacaaccg gccgaagcga gttttctcgg tgagaaggcc cgctttctcg agatggtcgc 2280
gaacctgatc gcgcaaagcg tcaattcgct cagggcgatc gagcgaaaac aagatcaatt 2340
gaccaacgaa cgcgattatc taaaacagga actagtcaaa aactaccgtt tcgaaaacat 2400
catcggccac tccgagccga tgctgaaggt attcgacatc atccggcaag tcgcgaaatg 2460
gaacacgacc gtgctgatac gcggcgagtc gggcaccggc aaagaagtcg tcgcgaacgc 2520
gatccatttc aattcagcct gcgcgaatgg cccgtttttg aaattgaatt gcgcggccct 2580
gcccgacacc ttgctcgaat ccgaactgtt cggccacgag aaaggcgcgt tcagcggcgc 2640
gatcggtcag cgcaaggggc gtttcgaact cgccgacaac ggcaccttgt ttctcgacga 2700
aatcggcgaa atttcggcct cgttccaagc caaattatta cgggtgctgc aagagggcga 2760
gttcgaacgt gtcggcggcg ttaaaacctt aaaagtgcat gtacgggtaa tcgccg 2816

Claims (10)

  1. The application of NifA protein or NifA coding gene or NifL protein coding gene in regulating and controlling methanotrophic bacteria to utilize nitrogen gas to carry out bacterial growth.
  2. Application of NifA protein or coding gene of NifA or NifL protein or coding gene of NifL protein in regulation and control of methanotrophic bacteria by using nitrogen as nitrogen source.
  3. The application of NifA protein or NifA coding gene or NifL protein coding gene in regulating methanotrophic bacteria for nitrogen fixation.
  4. The application of the coding gene of NifA protein or NifA or NifL protein in participating in the bacterial growth of methanotrophic bacteria by using nitrogen.
  5. 5. A method for determining whether a test protein in methanotrophic bacteria participates in the quantitative growth of methanotrophic bacteria by using nitrogen as a nitrogen source comprises the following steps:
    taking methanotrophic bacteria as spawn, and knocking out coding genes of tested proteins in the spawn through homologous recombination to obtain knock-out bacteria;
    respectively carrying out parallel culture on the spawn running bacteria and the knockout bacteria; if the time of entering the decay period of the growth curve of the knock-out bacteria is earlier than that of the emerging bacteria, the tested protein is a positive regulation protein participating in the bacterial quantity growth of the methanotrophic bacteria by using nitrogen as a nitrogen source; if the time of the growth curve of the knock-out bacteria entering the decay phase is the same as or later than that of the outgrowth bacteria and the mass peak value of the growth curve is higher than that of the outgrowth bacteria, the tested protein is a negative regulation protein participating in the growth of the methanotrophic bacteria by using nitrogen as a nitrogen source;
    the culture method comprises the following steps: culturing in a sealed environment by adopting a specific culture medium; the gas in the sealed environment is a mixed gas of nitrogen and oxygen, and the volume percentage of the oxygen in the mixed gas is 15-18%; the only nitrogen source in the specific medium is nitrate ion, and its concentration in the medium is 0.25-0.5 g.L-1。
  6. 6. A method for determining whether a test protein in methanotrophic bacteria participates in the quantitative growth of methanotrophic bacteria by using nitrogen as a nitrogen source comprises the following steps:
    taking methanotrophic bacteria as spawn, and knocking out coding genes of tested proteins in the spawn through homologous recombination to obtain knock-out bacteria;
    respectively carrying out parallel culture on the spawn running bacteria and the knockout bacteria; if the time of entering the decay period of the growth curve of the knock-out bacteria is earlier than that of the emerging bacteria, the tested protein is a positive regulation protein participating in the bacterial quantity growth of the methanotrophic bacteria by using nitrogen as a nitrogen source;
    the culture method comprises the following steps: culturing in a sealed environment by adopting a specific culture medium; the gas in the sealed environment is a mixed gas of nitrogen and oxygen, and the volume percentage of the oxygen in the mixed gas is 15-18%; the only nitrogen source in the specific medium was nitrate ion, and its concentration in the medium was 0.25-0.5g·L-1。
  7. 7. A method for determining whether a test protein in methanotrophic bacteria participates in the quantitative growth of methanotrophic bacteria by using nitrogen as a nitrogen source comprises the following steps:
    taking methanotrophic bacteria as spawn, and knocking out coding genes of tested proteins in the spawn through homologous recombination to obtain knock-out bacteria;
    respectively carrying out parallel culture on the spawn running bacteria and the knockout bacteria; if the time of the growth curve of the knock-out bacteria entering the decay phase is the same as or later than that of the outgrowth bacteria and the mass peak value of the growth curve is higher than that of the outgrowth bacteria, the tested protein is a negative regulation protein participating in the growth of the methanotrophic bacteria by using nitrogen as a nitrogen source;
    the culture method comprises the following steps: culturing in a sealed environment by adopting a specific culture medium; the gas in the sealed environment is a mixed gas of nitrogen and oxygen, and the volume percentage of the oxygen in the mixed gas is 15-18%; the only nitrogen source in the specific medium is nitrate ion, and its concentration in the medium is 0.25-0.5 g.L-1。
  8. 8. A method for determining whether a test protein in methanotrophic bacteria participates in the quantitative growth of methanotrophic bacteria by using nitrogen as a nitrogen source comprises the following steps:
    taking methanotrophic bacteria as spawn, and knocking out coding genes of tested proteins in the spawn through homologous recombination to obtain knock-out bacteria;
    culturing the knockout bacterium; if the growth curve of the knock-out bacteria enters a decline period within 72 hours of culture, the tested protein is a positive regulation protein participating in the growth of the methanotrophic bacteria by using nitrogen as a nitrogen source;
    the culture method comprises the following steps: culturing in a sealed environment by adopting a specific culture medium; the gas in the sealed environment is a mixed gas of nitrogen and oxygen, and the volume percentage of the oxygen in the mixed gas is 15-18%; the only nitrogen source in the specific medium is nitrate ion, and its concentration in the medium is 0.25-0.5 g.L-1。
  9. 9. The method of any of claims 5 to 8, wherein:
    knocking out the encoding gene of the test protein in the starting strain through homologous recombination is realized by introducing specific DNA molecules into the starting strain; the specific DNA molecule consists of an upstream homology arm, a resistance gene and a downstream homology arm; the upstream arm targets the upstream of the start codon of the coding gene of the test protein in the starting bacterium genome DNA, and the downstream arm targets the downstream of the stop codon of the coding gene of the test protein in the starting bacterium genome DNA.
  10. 10. A methanotrophic bacterium transformation method comprises the following steps:
    determining whether a test protein in methanotrophic bacteria is involved in the growth of methanotrophic bacteria using nitrogen as a nitrogen source using the method of any one of claims 5 to 9;
    and carrying out genetic engineering modification on the methanotrophic bacteria according to whether the test protein in the methanotrophic bacteria participates in the growth of the methanotrophic bacteria by using nitrogen as a nitrogen source.
CN202110507967.9A 2021-05-11 2021-05-11 Transformation and regulation method of nitrogen-fixing microorganisms Pending CN113248582A (en)

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Application publication date: 20210813