Artificial non-coding RNA molecule with ammonium transporter gene silencing function and application thereof

Abstract

The invention relates to an artificial non-coding RNA (Ribonucleic Acid) molecule with an ammonium transporter gene silencing function and application of the artificial non-coding RNA molecule. The invention provides an artificial non-coding RNA (Ribonucleic Acid) molecule, the RNA molecule is paired with a conserved complementary region of an ammonium transporter coding gene amtB promoter, and the nucleotide sequence of the RNA molecule is shown as SEQ ID NO: 1. The invention also provides an artificial combination module. The combination module sequentially comprises a promoter element for promoting gene expression under a high ammonium condition and a DNA molecule obtained by reverse transcription of the artificial non-coding RNA molecule in a direction from upstream to downstream. The invention further provides application of the RNA molecule in enhancing the ammonium secreting capacity of the nitrogen-fixing bacteria, expression of an ammonium transporter coding gene is inhibited by utilizing the gene coded by the artificial non-coding RNA molecule, and then the ammonium secreting capacity of the nitrogen-fixing bacteria is improved. The artificial combination module disclosed by the invention can obviously improve the ammonium secretion amount of chassis microorganisms, and the nitrogen fixation effect is improved.

Description

Technical Field

[0001] The present disclosure relates to the field of biotechnology, and in particular, to an artificial non-coding RNA molecule, a DNA molecule, a recombinant expression vector, a transformant, and application of the artificial non-coding RNA molecule in enhancing the ammonium secretion ability of nitrogen-fixing bacteria. Background Art

[0002] Biological nitrogen fixation is the process of nitrogen-fixing microorganisms reducing nitrogen in the air to ammonium through their own nitrogenase at normal temperature and pressure. Using biological nitrogen fixation to replace chemical nitrogen fertilizers can save fertilizer, increase production and increase efficiency, and has great application potential in agricultural production. However, due to the influence of natural defects such as "high ammonium does not fix nitrogen, and nitrogen fixation does not secrete ammonium", the application of biological nitrogen fixation in agriculture is greatly limited. Therefore, the construction of efficient ammonium secretion engineering strains is currently a hot topic in biological nitrogen fixation research.

[0003] Ammonium transporter AmtB is a protein that transports NH4 + The protein can transport extracellular ammonium into the cell as its own nitrogen source, ensuring the stability of intracellular ammonium and the normal operation of nitrogen metabolism. Therefore, knocking out the gene of ammonium active transport protein AmtB and reducing the reflux of excreted ammonium is one of the main strategies for constructing ammonium-secreting engineering strains.

[0004] Artificial non-coding RNA molecules can quickly and effectively regulate the expression of specific single or multiple target genes without changing chromosomal genes. Therefore, by designing artificial non-coding RNA molecules with the ability to silence the gene of the ammonium active transport protein AmtB and transferring it into the nitrogen-fixing microbial chassis, the ammonium secretion capacity of nitrogen-fixing bacteria can be enhanced, which is of great significance for the construction of an efficient rhizosphere nitrogen fixation system. Summary of the invention

[0005] The purpose of the present disclosure is to design an artificial non-coding RNA functional module with the ability to enhance the ammonium secretion of nitrogen-fixing bacteria, so as to construct an efficient ammonium-secreting engineered bacteria, and then to construct an efficient nitrogen fixation system, so as to further meet the needs of practical applications.

[0006] In order to achieve the above-mentioned object, the first aspect of the present disclosure provides an artificial non-coding RNA molecule, which is paired with a conservative complementary region of the promoter of a gene encoding an ammonium transporter, and the nucleotide sequence of the RNA molecule is shown in SEQ ID NO: 1.

[0007] The second aspect of the present disclosure provides a DNA molecule, which is transcribed into the RNA molecule described in the first aspect.

[0008] The third aspect of the present disclosure provides an artificial combination module, which comprises, in order from upstream to downstream, a promoter element for initiating transcription of the DNA molecule of the second aspect and the DNA molecule.

[0009] Optionally, the nucleotide sequence of the promoter element is shown as SEQ ID NO: 2; and the nucleotide sequence of the artificial combination module is shown as SEQ ID NO: 3.

[0010] The fourth aspect of the present disclosure provides a recombinant expression vector, into which the artificial combination module described in the third aspect is inserted, and an expression frame for regulating the expression of a gene encoding an ammonium transporter is formed.

[0011] A fifth aspect of the present disclosure provides a transformant, into which the recombinant expression vector described in the fourth aspect is introduced.

[0012] Optionally, the host cell of the transformant is a nitrogen-fixing bacterium.

[0013] Optionally, the nitrogen-fixing bacteria include: Pseudomonas stutzeri P. stutzeri A1501 or Klebsiella pneumoniae K. pneumoniae Azotobacter vinelandii A.vinelandii .

[0014] The sixth aspect of the present disclosure provides the use of the RNA molecule described in the first aspect in enhancing the ammonium secretion ability of nitrogen-fixing bacteria, using the gene encoded by the artificial non-coding RNA molecule to inhibit the expression of the gene encoding the ammonium transporter to enhance the ammonium secretion ability of nitrogen-fixing bacteria.

[0015] Optionally, the application method is to allow the transformant containing the gene encoded by the artificial non-coding RNA molecule to bind to the root surface of the host crop or invade the inside of the root system to form a rhizosphere nitrogen fixation system with the host crop; the host crop includes corn or rice.

[0016] Through the above technical scheme, the present invention uses synthetic biology methods to design an artificial non-coding RNA molecule, and then constructs a recombinant expression vector pLAFR3-AnsR-BU2 containing an ammonium secretion module, and the recombinant expression vector is respectively transferred into different nitrogen-fixing microbial chassis to obtain three recombinant nitrogen-fixing engineering bacteria: A1501-AnsR-BU2, K.pneumonia -AnsR-BU2, A.vinelandii -AnsR-BU2. The introduction of the combined module AnsR-BU2 of the artificial non-coding RNA molecule encoding gene disclosed in the present invention will not affect the growth of chassis microorganisms, and can significantly enhance the ammonium secretion ability of nitrogen-fixing bacteria and improve the nitrogen fixation effect by inhibiting and regulating the expression of the ammonium transporter encoding gene.

[0017] Other features and advantages of the present disclosure will be described in detail in the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The accompanying drawings are used to provide a further understanding of the present disclosure and constitute a part of the specification. Together with the following specific embodiments, they are used to explain the present disclosure but do not constitute a limitation of the present disclosure. In the accompanying drawings: Figure 1 Schematic diagram of the construction of the recombinant expression vector of the combined module AnsR-BU2 encoding artificial non-coding RNA gene, with the insertion site being Bam HI and Hin d Ⅲ.

[0019] Figure 2 This is the positive colony PCR verification result of the recombinant expression vector pLAFR3-AnsR-BU2 and the recombinant nitrogen-fixing bacteria introduced into it.

[0020] Figure 3 Growth curves of chassis nitrogen-fixing strains and recombinant engineered bacteria.

[0021] Figure 4 This is a graph showing the results of the ammonium secretion capacity test of chassis nitrogen-fixing strains and recombinant engineered bacteria. DETAILED DESCRIPTION

[0022] The specific implementation of the present disclosure is described in detail below in conjunction with the accompanying drawings. It should be understood that the specific implementation described herein is only used to illustrate and explain the present disclosure, and is not used to limit the present disclosure.

[0023] In a first aspect, the present disclosure provides an artificial non-coding RNA molecule, which pairs with a conservative complementary region of a promoter of a gene encoding an ammonium transporter, and the nucleotide sequence of the RNA molecule is shown in SEQ ID NO: 1.

[0024] In the sequence of SEQ ID NO: 1, the nucleotide symbol "t" is used to represent "u".

[0025] In the present disclosure, the sequence of the artificial non-coding RNA molecule is located in the complementary pairing region of the conservative sequence of the promoter of the ammonium transporter AmtB gene, and the artificial non-coding RNA molecule is completely or incompletely complementary to the 5'UTR of the target gene mRNA, thereby inhibiting the translation of the mRNA, and finally regulating the expression of the target gene at the post-transcriptional level. The sequence of the artificial non-coding RNA molecule can be a conservative sequence of the promoter region of the nitrogen fixation-related gene obtained by bioinformatics analysis of the promoter sequences of nitrogen fixation-related genes of Pseudomonas stutzeri, Klebsiella pneumoniae and Azotobacter vinelandii, and an artificial non-coding RNA molecule containing a complementary pairing region of the conservative sequence of the promoter of the nitrogen fixation-related gene is synthesized by an artificial chemical synthesis method.

[0026] The second aspect of the present disclosure provides a DNA molecule, which is transcribed into the RNA molecule described in the first aspect.

[0027] In the present disclosure, the DNA molecule is obtained by reverse transcription from the artificial non-coding RNA molecule described in the first aspect of the present disclosure.

[0028] The third aspect of the present disclosure provides an artificial combination module, which comprises, in order from upstream to downstream, a promoter element for initiating transcription of the DNA molecule of the second aspect and the DNA molecule.

[0029] The terms "upstream" and "downstream" in the present disclosure refer to upstream and downstream with respect to the expression direction of a gene.

[0030] According to the present disclosure, the nucleotide sequence of the promoter element is shown as SEQ ID NO: 2; the nucleotide sequence of the artificial combination module is shown as SEQ ID NO: 3.

[0031] The artificial combination module disclosed in the present invention is named AnsR-BU2. The transcription of the DNA molecule is controlled by a promoter element that initiates gene expression under high ammonium conditions. The full length of the AnsR-BU2 fragment is 546 bp.

[0032] The fourth aspect of the present disclosure provides a recombinant expression vector, into which the artificial combination module described in the third aspect is inserted, and an expression frame for regulating the expression of a gene encoding an ammonium transporter is formed.

[0033] In the present disclosure, the vector used to construct the recombinant expression vector is the broad-host expression vector pLAFR3. Bam HI and Hind III, double enzyme digestion was performed on it, homology arms were added to the fragment of the artificially synthesized ammonium secretion regulatory module AnsR-BU2 during primer design, and the artificially synthesized ammonium secretion regulatory module AnsR-BU2 fragment was inserted into its multiple cloning site by seamless cloning to obtain the disclosed Figure 1 The recombinant expression vector pLAFR3-AnsR-BU2 containing the artificial non-coding RNA encoding gene is shown.

[0034] A fifth aspect of the present disclosure provides a transformant, into which the recombinant expression vector described in the fourth aspect is introduced.

[0035] According to the present disclosure, the host cell of the transformant is a nitrogen-fixing bacterium. Preferably, the nitrogen-fixing bacterium includes: Pseudomonas stutzeri P. stutzeri A1501 or Klebsiella pneumoniae K. pneumoniae Azotobacter vinelandii A.vinelandii .

[0036] In the present disclosure, the inventors of the present disclosure respectively transferred the transformants into three different nitrogen-fixing microbial chassis: nitrogen-fixing Pseudomonas stutzeri P. stutzeriA1501, Klebsiella pneumoniae K. pneumoniae Azotobacter vinelandii A.vinelandii In this study, three recombinant nitrogen-fixing engineering strains A1501-AnsR-BU2, K. pneumoniae -AnsR-BU2, A.vinelandii -AnsR-BU2, the PCR results of the nitrogen-fixing engineering bacteria positive colonies were as follows Figure 2 As shown, band 1 is a negative control; bands 2-4 are the amplification results of the artificially synthesized ammonium secretion regulatory module AnsR-BU2 fragment in three recombinant nitrogen-fixing engineering bacteria.

[0037] The sixth aspect of the present disclosure provides the use of the RNA molecule described in the first aspect in enhancing the ammonium secretion ability of nitrogen-fixing bacteria, using the gene encoded by the artificial non-coding RNA molecule to inhibit the expression of the gene encoding the ammonium transporter to enhance the ammonium secretion ability of nitrogen-fixing bacteria.

[0038] In one embodiment of the present disclosure, the application method is to allow the transformant containing the gene encoded by the artificial non-coding RNA molecule to bind to the root surface of the host crop or invade the inside of the root system to form a rhizosphere nitrogen fixation system with the host crop; the host crop includes corn or rice.

[0039] The expression of the recombinant expression vector pLAFR3-AnsR-BU2 disclosed in the present invention is controlled by an inducible promoter that is highly expressed under high ammonium conditions. In nitrogen-fixing engineering bacteria, the ammonium secretion of nitrogen-fixing bacteria is increased by promoting the expression of nitrogen-fixing related genes. The present invention proves that the combination module of the artificial non-coding RNA molecule encoding gene disclosed in the present invention can significantly improve the ammonium secretion ability of nitrogen-fixing bacteria without affecting the growth of chassis nitrogen-fixing bacteria by measuring the growth curve and ammonium secretion of the chassis nitrogen-fixing bacteria transferred into the AnsR-BU2 ammonium secretion regulatory module, and the ammonium secretion amount can be increased by more than 1.5 times. The specific results are as follows Figure 3 and Figure 4 shown.

[0040] The present disclosure is further described in detail below through examples, but the present disclosure is not limited thereby.

[0041] In the following examples, where no specific experimental conditions are specified, they are based on conventional conditions familiar to those skilled in the art, such as the conditions described in "Sambrook" et al. Molecular Cloning: A Reference Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or the conditions recommended by the manufacturer.

[0042] Example 1 This example is used to illustrate the design and synthesis of the combination module AnsR-BU2 of artificial non-coding RNA encoding genes.

[0043] The conserved sequence of the promoter region of nitrogen-fixing related genes was obtained by bioinformatics analysis of the promoter sequences of nitrogen-fixing Pseudomonas stutzeri, Klebsiella pneumoniae and Azotobacter vinelandii, and an artificial non-coding RNA molecule complementary to the conserved sequence of the promoter of the ammonium transporter gene was synthesized by an artificial chemical synthesis method, and its expression was controlled by a promoter with high expression under high ammonium conditions. The coding DNA molecule was obtained by reverse transcription of the artificially synthesized non-coding RNA molecule, and the DNA molecule was combined with a promoter element with high expression under high ammonium conditions that initiated the transcription of the DNA molecule to obtain a combined module AnsR-BU2 of an artificial non-coding RNA coding gene with a total length of 546 bp, and its nucleotide sequence is shown in SEQ ID NO: 1, wherein the nucleotide symbol "t" represents "u".

[0044] Example 2 This example is used to illustrate the construction of nitrogen-fixing engineering bacteria containing the AnsR-BU2 module.

[0045] (1) Construction of recombinant expression vector: like Figure 1 As shown, the wide host expression vector pLAFR3 was Bam HI and Hind III double enzyme digestion, and insert the artificial chemically synthesized non-coding RNA molecule into the multiple cloning site of pLAFR3 through seamless cloning technology. The obtained positive strain is subjected to PCR sequencing to verify the correctness of the sequence, and the recombinant expression vector pLAFR3-AnsR-BU2 containing the artificial non-coding RNA encoding gene disclosed in the present invention is obtained.

[0046] (2) Construction of recombinant nitrogen-fixing engineering bacteria: The obtained recombinant expression vector plasmid was transferred into three different nitrogen-fixing microorganisms (Pseudomonas stutzeri) by triparental conjugation or electroporation. P. stutzeri A1501, Klebsiella pneumoniae K. pneumoniae Azotobacter vinelandii A. vinelandii ) successfully constructed a recombinant nitrogen-fixing engineering bacteria containing the expression vector pLAFR3-AnsR-BU2 containing an artificial non-coding RNA coding gene combination module. The three recombinant nitrogen-fixing engineering bacteria obtained were: A1501-AnsR-BU2, K. pneumoniae -AnsR-BU2, A.vinelandii -AnsR-BU2, the positive colony PCR results were as follows Figure 2 As shown, bands 2-4 are the amplification results of the artificially synthesized ammonium secretion regulatory module AnsR-BU2 fragment in three recombinant nitrogen-fixing engineering bacteria, indicating that the recombinant nitrogen-fixing engineering bacteria were successfully constructed.

[0047] Example 3 This example is used to illustrate the determination of the growth curves of recombinant nitrogen-fixing engineering bacteria and chassis bacteria.

[0048] This embodiment includes the following methods: (1) Select the recombinant nitrogen-fixing bacteria A1501-AnsR-BU2, K. pneumoniae -AnsR-BU2, A.vinelandii -AnsR-BU2 and wild-type single colonies were inoculated into LB medium (the recombinant bacteria may be resistant to tetracycline (Tc), while the wild-type is not resistant), and the culture conditions were 30°C and shaken at 200 rpm overnight.

[0049] (2) Centrifuge the overnight culture at 4°C, 5000 rpm for 10 min, remove the supernatant, and recover the bacteria.

[0050] (3) Wash the cells twice with 20 mL of 0.85% NaCl solution, then resuspend the cells in the corresponding liquid culture medium. The OD of each bacterial suspension is 600 Adjust the value to 1.0.

[0051] (4) Add 2 mL of bacterial suspension and 18 mL of liquid culture medium to the flask. 600 The value was adjusted to 0.1. 200 μL of bacterial solution was added to each well of a 96-well plate. Three parallels were set for each strain. The plate was placed in a Bioscreen C growth curve analyzer and the OD was measured every 2 hours. 600 value.

[0052] The results are as follows Figure 3 As shown in the figure, it is proved that the growth curves of the recombinant nitrogen-fixing engineering bacteria and the chassis microorganisms are basically the same. After 20 hours of cultivation, they entered the stable phase, and the OD 600 The values ​​are all around 0.8, indicating that the introduction of the combination module AnsR-BU2 of the artificial non-coding RNA molecule encoding gene will not affect the normal growth of chassis microorganisms.

[0053] Example 4 This example is used to illustrate the determination of the ammonium secretion ability of recombinant nitrogen-fixing engineering bacteria under nitrogen-fixing conditions.

[0054] This embodiment includes the following methods: 1. NH4 + Color developer preparation.

[0055] Determine the ammonium ion concentration in the culture medium using an ELISA instrument: prepare 100 mL of 0.5 mM phosphate buffer (pH 6.8) (51.0 mL of 0.5 mM NaH2PO4 +, 49.0mL, 0.5 mM Na2HPO4), add o-phthalaldehyde (0.201g o-phthalaldehyde per 100mL buffer) and 50mM β-mercaptoethanol (0.352mL β-mercaptoethanol per 100mL buffer) to the buffer to a final concentration of 15mM. Store at 4°C in the dark.

[0056] 2. Determination of the ammonium secretion ability of the strain.

[0057] (1) Activate the strain on an antibiotic resistance screening plate and inoculate the activated strain into 20 mL of liquid LB medium containing antibiotics at 30°C and shake at 220 rpm overnight.

[0058] (2) Transfer 20 mL of overnight culture solution to a centrifuge tube and centrifuge at 4°C, 5500 rpm for 8 min to collect the cells.

[0059] (3) Add 20 mL of saline to the centrifuge tube to fully suspend the precipitated bacteria and adjust the concentration of the bacterial solution to OD 600 The value is 1.0.

[0060] (4) P. stutzeri The bacterial suspensions of A1501 and recombinant nitrogen-fixing engineering bacteria A1501-AnsR-BU2 were transferred to ground-mouth Erlenmeyer flasks containing LB medium (without N) containing Km, and the OD 600 It can be adjusted to 0.1 and plugged with a rubber stopper; Will A. vinelandii and recombinant nitrogen-fixing bacteria A.vinelandii -AnsR-BU2 bacterial suspensions were transferred to ground-mouth Erlenmeyer flasks containing Buik's medium (without N), and the OD 600 Adjust to 0.1 and plug in the rubber stopper; Will K. pneumoniae and recombinant nitrogen-fixing bacteria K. pneumoniae -AnsR-BU2 bacterial suspensions were transferred into ground-mouth Erlenmeyer flasks containing sucrose-containing minimal medium (without N) and nitrogen gas, and the OD 600 Adjust to 0.1 and plug in the rubber stopper.

[0061] (5) Use the inlet needle to pierce the rubber stopper and continue to fill the sealed culture bottle with pure nitrogen. At the same time, exhaust the air in the bottle through the outlet needle. After five minutes, pull out the inlet needle first and then the outlet needle.

[0062] (6) Use a syringe to inject 1% (1 mL) of pure oxygen into the culture bottle to fill the remaining volume (excluding the volume of the culture solution) in the bottle. The culture conditions are 30°C and continuous culture with shaking at 220 rpm.

[0063] (7) Starting from the 4th hour, use a microsyringe to take 1 mL of culture medium from the culture bottle every 4 hours and inject it into a 1.5 mL centrifuge tube. Centrifuge at 12,000 × g for 10 min and use a pipette to transfer 200 µL of the supernatant to a new 1.5 mL centrifuge tube.

[0064] (8) Add 50 μL of colorimetric reagent to 200 μL of the supernatant after centrifugation, incubate at 63°C for 10 min, cool to room temperature, and transfer all the reaction solution to a 96-well plate (black) using a pipette. This process must be kept away from light (covered with aluminum foil).

[0065] (9) The sample can be measured on the microplate reader under the conditions of 410nm excitation light and 470nm emission light.

[0066] 3. Draw NH4 + Standard curve.

[0067] (1) First, prepare a 100 μM NH4Cl standard solution (stock solution). On this basis, dilute the standard solutions to concentrations of 20 μM, 10 μM, 5 μM, 2.5 μM, and 1.25 μM, respectively, and use sterile double distilled water as a control. Add 200 μL of the standard solution to a 1.5 mL centrifuge tube, and then add 50 μL of the color developer. Incubate at 63 °C for 10 min, cool to room temperature, and transfer the reaction solution to a 96-well plate with a pipette. Keep the reaction away from light (cover with aluminum foil).

[0068] (2) The samples were measured under the conditions of 410nm excitation light and 470nm emission light.

[0069] (3) The software can automatically generate colorimetric data in Excel form. After repeated iterations, NH4 + Content standard curve.

[0070] (4) To ensure the parallelism and reliability of the test, the standard curve must be measured each time the ammonium concentration of the sample is measured.

[0071] The results are as follows Figure 4 As shown, it is proved that the chassis nitrogen-fixing bacteria P. stutzeri A1501, K. pneumoniae and A.vinelandii In comparison, the ammonium secretion capacity of the three recombinant nitrogen fixation engineering strains was significantly improved, by 2 times, 1.5 times and 1.5 times respectively; indicating that the combination module AnsR-BU2 of artificial non-coding RNA molecule encoding genes can significantly improve the ammonium secretion capacity of the chassis strain.

[0072] The preferred embodiments of the present disclosure are described in detail above in conjunction with the accompanying drawings; however, the present disclosure is not limited to the specific details in the above embodiments. Within the technical concept of the present disclosure, a variety of simple modifications can be made to the technical solution of the present disclosure, and these simple modifications all fall within the protection scope of the present disclosure.

[0073] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present disclosure will not further describe various possible combinations.

[0074] In addition, various embodiments of the present disclosure may be arbitrarily combined, and as long as they do not violate the concept of the present disclosure, they should also be regarded as the contents disclosed by the present disclosure.

Claims

1. An artificial non-coding RNA molecule, characterized in that The RNA molecule is paired with the conservative complementary region of the promoter of the ammonium transport protein encoding gene, and the nucleotide sequence of the RNA molecule is shown as SEQ ID NO:

1.

2. A DNA molecule, characterized in that The DNA molecule is transcribed to obtain the RNA molecule according to claim 1.

3. An artificial combination module, characterized in that: The combined module includes, in order from upstream to downstream, a promoter element for initiating transcription of the DNA molecule of claim 2 and the DNA molecule.

4. The artificial combination module according to claim 3, wherein: The nucleotide sequence of the promoter element is shown in SEQ ID NO: 2; The nucleotide sequence of the artificial combination module is shown in SEQ ID NO:

3.

5. A recombinant expression vector, characterized in that: The recombinant expression vector is inserted with the artificial combination module according to claim 3 or 4, and forms an expression frame for regulating the expression of the ammonium transport protein encoding gene.

6. A transformant, characterized in that: The recombinant expression vector according to claim 5 is introduced into the transformant.

7. The transformant according to claim 6, wherein The host cell of the transformant is a nitrogen-fixing bacterium.

8. The transformant according to claim 7, wherein The nitrogen-fixing bacteria include: Pseudomonas stutzeri P. stutzeri A1501 or Klebsiella pneumoniae K. pneumoniae Azotobacter vinelandii A.vinelandii .

9. Use of the RNA molecule according to claim 1 in enhancing the ammonium secretion ability of nitrogen-fixing bacteria, characterized in that: The gene encoded by the artificial non-coding RNA molecule is used to inhibit the expression of the gene encoding the ammonium transporter, thereby enhancing the ammonium secretion ability of the nitrogen-fixing bacteria.

10. The use according to claim 9, wherein: The application method is to make the transformant containing the gene encoded by the artificial non-coding RNA molecule bind to the root surface of the host crop or invade the inside of the root system to form a rhizosphere nitrogen fixation system with the host crop; the host crop includes corn or rice.

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