Nitrogen-fixing cyanobacteria heteroplasm as well as rapid induction method and application thereof

Abstract

The invention relates to a nitrogen-fixing cyanobacteria heteromorphic cell and a rapid induction method and application thereof, and the rapid induction comprises the following steps: putting nitrogen-fixing cyanobacteria in a nitrogen-deficient culture solution under an illumination condition, inducing for 12 hours, then putting the taken nitrogen-fixing cyanobacteria in a nitrogen-deficient phosphorus-supplemented culture solution, and continuously inducing for 12 hours; meanwhile, a high-content cell imaging analysis system is used for quantifying the quantity, the cell size and the relative fluorescence intensity of the heteromorphic cells formed by differentiation; compared with induction in a culture solution which is deficient in nitrogen and not supplemented with phosphorus, the number of the heteromorphic cells of the three nitrogen-fixing blue-green algae is averagely increased by 43.3%, and the number of the heteromorphic cells of the anabaena is increased by 116%; the culture mode of nitrogen deficiency and phosphorus supply can accelerate the process of inducing heteromorphic cell differentiation, and finds that the number of the heteromorphic cells differentiated from the three strains has the best effect under the conditions of 24 hours of induction in two steps and three times of phosphorus supply.

Description

Technical Field

[0001] The invention belongs to the field of nitrogen-fixing cyanobacteria heterocysts, and in particular relates to nitrogen-fixing cyanobacteria heterocysts and a rapid induction method and application thereof. Background Art

[0002] Nitrogen-fixing cyanobacteria is a general term for cyanobacteria that have the ability to fix nitrogen. Anabaenasp. )、Nostoc ( Nostocsp. ) and Aphanizomenon ( Aphanizomenonsp. ). All three algae belong to the Cyanophyta, Cyanophyceae, Nostocales, and Nostocaceae. They can utilize various forms of nitrogen sources in the environment, such as ammonium salts, nitrates, and nitrogen gas. When utilizing combined nitrogen sources such as ammonium salts and nitrates, the entire hyphae is composed of cells of a single form, which is the site of photosynthesis and is called a vegetative cell. Under the conditions of lack of nitrate or ammonium induction, about 5%-10% of the vegetative cells on the hyphae of the three algae can differentiate into a type of cell that is relatively special in morphology and function, namely, a heterocyst. Under aerobic conditions, the vegetative cells of cyanobacteria can perform photosynthesis normally to release oxygen. Since nitrogenase is inactivated when it encounters oxygen, oxygenic photosynthesis and nitrogen fixation are incompatible processes. The heterocysts differentiate into thick-walled structures to reduce oxygen penetration, thereby protecting the internal nitrogenase from being destroyed by oxygen and maintaining its activity. Nitrogenase can convert nitrogen into ammonia in the heterocysts and transfer ammonia to the nutrient cells through intercellular filaments to complete the nitrogen fixation activity. Heterocysts are specialized cells for nitrogen fixation in cyanobacteria, usually formed when nitrogen is scarce in the environment. The three cyanobacteria in this patent are all cyanobacteria that can differentiate heterocysts for nitrogen fixation. The nitrogen fixation efficiency of the cyanobacteria is directly related to the characteristics of the heterocysts. The number, size and internal nitrogenase activity of the heterocysts are all direct factors that determine the nitrogen fixation efficiency of the cyanobacteria. At present, the induction of heterocysts mostly stays in the optimization of the induction process by changing environmental factors such as light intensity, light-dark cycle conditions, salt concentration, etc. The induction of heterocysts has not yet been promoted and applied on a large scale. The reason may be related to the induction speed of the current method. With the continuous deepening of research, more strategies to improve the induction of heterocysts with the idea of ​​optimizing nutritional conditions may be developed in the future. Summary of the invention

[0003] The present invention adopts a high-content cell imaging analysis system to quantify the heterocysts of nitrogen-fixing cyanobacteria, and obtains a rapid induction method for heterocysts of nitrogen-fixing cyanobacteria through algorithm analysis, providing a new option for the application of heterocysts in biotechnology.

[0004] The purpose of the present invention is achieved through the following technical solutions: A method for rapidly inducing heterocysts of nitrogen-fixing cyanobacteria comprises the following steps: Step 1: Place nitrogen-fixing cyanobacteria in nitrogen-deficient culture medium BG11-N under light conditions. 0 Medium induction 12h; Step 2: Take out the nitrogen-fixing cyanobacteria induced in step 1 and place it in the nitrogen-deficient and phosphorus-supplemented culture medium BG11-N 0 Induction was continued for 12 h in P; The concentration ratio of the phosphorus source in the culture solution of step one and step two is 1:(2-3).

[0005] As a more optimal technical solution of the present invention: the concentration ratio of the phosphorus source in the culture solution of step one and step two is 1:3.

[0006] As a more optimal technical solution of the present invention: the nitrogen-deficient culture solution lacks nitrogen sources in its components compared with the BG11 culture solution.

[0007] As a more optimal technical solution of the present invention: the phosphorus source in the nitrogen-deficient and phosphorus-supplemented culture solution and the nitrogen-deficient culture solution is dipotassium hydrogen phosphate.

[0008] As a more optimal technical solution of the present invention: the concentration of dipotassium hydrogen phosphate in the nitrogen-deficient culture medium in step one is 40 mg / L, and the concentration of dipotassium hydrogen phosphate in the nitrogen-deficient and phosphorus-supplemented culture medium in step two is 80-120 mg / L.

[0009] As a more optimal technical solution of the present invention: the induction time of step 1 and step 2 is 12 hours, and the total induction time is 24 hours.

[0010] As a more preferred technical solution of the present invention: the nitrogen-fixing cyanobacteria are Anabaena, Nostoc or Aphanizomenon.

[0011] As a more optimal technical solution of the present invention: the intensity of the light is 1000 Lux, the induced temperature is 20-25° C., and the shaking culture speed is 120 r / min.

[0012] Another object of the present invention is to provide a nitrogen-fixing cyanobacterial heterocyst, wherein the heterocyst is prepared by the above-mentioned nitrogen-fixing cyanobacterial heterocyst rapid induction method.

[0013] Another object of the present invention is to provide the use of the nitrogen-fixing cyanobacteria heterocystis in the preparation of biological nitrogen fertilizer.

[0014] The beneficial effects are as follows: The present invention uses a high-content cell imaging analysis system to quantify the heterocysts of nitrogen-fixing cyanobacteria, and obtains a rapid induction method for heterocysts of nitrogen-fixing cyanobacteria through algorithm analysis. Compared with the conventional induction method (in the nitrogen-deficient culture medium BG11-N 0 Induction), the present invention uses a specific phosphorus source concentration (BG11-N0 P 2 The phosphorus source addition amount is BG11-N 0 2 times, BG11-N 0 P 3 The amount of phosphorus source added is BG11-N 0 3 times of that of the culture time) and promoted the formation of heterocysts of nitrogen-fixing cyanobacteria, accelerating the nitrogen fixation process of nitrogen-fixing cyanobacteria.

[0015] Proven BG11-N 0 Induce three algae heterocysts in the culture medium for 12 hours, take them out and put them into BG11-N 0 P 2 and BG11-N 0 P 3 Induction was continued in the culture medium for 12 h, and BG11-N 0 P 3 The culture medium is most conducive to the development of heterocysts and shortens the induction period.

[0016] The phosphorus source used in the present invention can be dipotassium hydrogen phosphate, which is low in price and easy to popularize and apply, without the need to use large-scale instruments and equipment and dangerous devices. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] FIG1 is a diagram of heteromorphic cells of three algae obtained in Examples 1, 3, 4 and 5 observed under high-content imaging, wherein a, b and c represent the heteromorphic cells of Anabaena, Nostoc and Aphanizomenon in BG11, BG11-N, respectively. 0 、BG11-N 0 P 2 and BG11-N 0 P 3 Cultivated form.

[0018] Figure 2 The cell sizes and relative fluorescence intensities of the heteromorphic cells differentiated by the three algae obtained in Examples 1, 3, 4 and 5 under different culture conditions are shown in Table 1. BG11 (sampled after Example 1), BG11-N 0 (sampled after Example 3), BG11-N 0 P 2 (sampled after Example 4) and BG11-N 0 P 3 (Sampling after Example 5).

[0019] Figure 3 shows the heteromorphic cells of three algae obtained in Example 3, Example 6 and Example 7 observed under high-content imaging. Figures a, b and c represent Anabaena, Nostoc and Aphanizomenon in BG11-N 0 (sampled after Example 3), BG11-N 0 P2 (sampled after Example 6) and BG11-N 0 P 3 (Sampling after Example 7).

[0020] Figure 4 The cell sizes and relative fluorescence intensities of heteromorphic cells differentiated from the three algae obtained in Examples 3, 6 and 7 under different culture conditions are shown in Figure 1. 0 (Sampling after Example 3) BG11-N 0 P 2 (sampled after Example 6) and BG11-N 0 P 3 (Sampling after Example 7).

[0021] FIG5 is a picture of heteromorphic cells of three algae obtained in Example 10, Example 8 and Example 9 observed under high-content imaging, a, b and c represent Anabaena, Nostoc and Aphanizomenon, respectively. 0 (sampled after Example 10), BG11-N 0 P 2 (sampled after Example 8) and BG11-N 0 P 3 (Sampling after Example 9).

[0022] Figure 6 The cell sizes and relative fluorescence intensities of heteromorphic cells differentiated from the three algae obtained in Example 10, Example 8 and Example 9 under different culture conditions.

[0023] Figure 7 These are the microscope images of three algae obtained in Example 1, Example 3, Example 4 and Example 5, where a, b, c and d represent BG11, BG11-N respectively. 0 、BG11-N 0 P 2 and BG11-N 0 P 3 Microscope image of algae in the culture medium. From left to right, they are Anabaena, Nostoc, Aphanizomenon, BG11 (sampled after Example 1), BG11-N 0 (sampled after Example 3), BG11-N 0 P 2 (sampled after Example 4) and BG11-N 0 P 3 (Sampling after Example 5).

[0024] Figure 8Channel images and overlay images of TL10, TexasRed, and CHLA for observing differentiated heterocysts of nitrogen-fixing cyanobacteria using high-content imaging (heterocysts are marked in blue after overlay). DETAILED DESCRIPTION

[0025] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the art or the product instructions are used. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased through regular channels.

[0026] Example 1 Inoculate Anabaena, Nostoc or Aphanizomenon into 500 mL Erlenmeyer flasks filled with BG11 medium, seal the flasks with film, and place them on a constant temperature shaker at 120 rpm, 1000 Lux, and 25 °C for shaking culture. Determine the OD 750 The components and contents of the BG11 medium are shown in Table 1.

[0027] Table 1 The BG11 culture medium preparation method comprises weighing various raw materials according to the ratio, mixing and sterilizing according to the conventional method.

[0028] Example 2 Take 30 mL of each of the three algae strains prepared with the BG11 medium in Example 1, centrifuge at 4000 r / min for 2 min, collect the cells, and add them to the BG11-N medium. 0 The cells were resuspended in the culture medium, and the centrifugation and resuspending operations were repeated twice. The bottle mouth was sealed with a film and placed on a constant temperature shaker at 120 rpm, 1000 Lux, 25°C, and shaken for 12 hours. 0 The culture medium includes the following components and their contents are shown in Table 2.

[0029] Table 2 The BG11-N 0 Method for preparing culture medium: weigh each raw material according to the ratio, mix and sterilize according to the conventional method.

[0030] Example 3 Take 30 mL of each of the three algae strains prepared with the BG11 medium in Example 1, centrifuge at 4000 r / min for 2 min, collect the cells, and add them to the BG11-N medium. 0Resuspend the bacteria in the culture medium, repeat the centrifugation and resuspending operation twice, cover the bottle mouth with sealing film and place it on a constant temperature shaker at 120 rpm, 1000 Lux, 25°C, and culture for 24 hours.

[0031] The BG11-N 0 Method for preparing culture medium: weigh each raw material according to the ratio, mix and sterilize according to the conventional method.

[0032] Example 4 Take 30 mL of each of the three algae cultured in the BG11 medium in Example 1, centrifuge at 4000 r / min for 2 min, collect the cells, and add them to the BG11-N medium. 0 P 2 The cells were resuspended in the culture medium, and the centrifugation and resuspending operations were repeated twice. The bottle mouth was sealed with a film and placed on a constant temperature shaker at 120 rpm, 1000 Lux, 25°C, and shaken for 24 hours. 0 P 2 The culture medium includes the following components and their contents are shown in Table 3.

[0033] Table 3 The BG11-N 0 P 2 Preparation method of culture medium: weigh each raw material according to the above ratio, mix and sterilize according to the conventional method.

[0034] Example 5 Take 30 mL of each of the three algae cultured in the BG11 medium in Example 1, centrifuge at 4000 r / min for 2 min, collect the cells, and add them to the BG11-N medium. 0 P 3 The cells were resuspended in the culture medium, and the centrifugation and resuspending operations were repeated twice. The bottle mouth was sealed with a film and placed on a constant temperature shaker at 120 rpm, 1000 Lux, 25°C, and shaken for 24 hours. 0 P 3 The culture medium includes the following components and their contents are shown in Table 4.

[0035] Table 4 The BG11-N 0 P 3 Preparation method of culture medium: weigh each raw material according to the above ratio, mix and sterilize according to the conventional method.

[0036] Example 6 Take 30 mL of each of the three strains of cyanobacteria cultured in Example 2, centrifuge at 4000 r / min for 2 min, collect the cells, and place them in BG11-N 0 P 2 The culture was cultured in the culture medium for 24 hours, and a sealing film was added to the bottle mouth. The bottle was then placed on a constant temperature shaker at 120 rpm, 1000 Lux, and 25°C for shaking culture. Samples were taken at the 25th hour.

[0037] Example 7 Take 30 mL of each of the three strains of cyanobacteria cultured in Example 2, centrifuge at 4000 r / min for 2 min, collect the cells, and place them in BG11-N 0 P 3 The culture was cultured in the culture medium for 24 hours, and a sealing film was added to the bottle mouth. The bottle was then placed on a constant temperature shaker at 120 rpm, 1000 Lux, and 25°C for shaking culture. Samples were taken at the 25th hour.

[0038] Example 8 Take 30 mL of each of the three strains of cyanobacteria cultured in Example 3, centrifuge at 4000 r / min for 2 min, collect the cells, and place them in BG11-N 0 P 2 The culture was cultured in the culture medium for 48 hours, and a sealing film was added to the bottle mouth. The bottle was then placed on a constant temperature shaker at 120 rpm, 1000 Lux, and 25°C for shaking culture. Samples were taken at the 49th hour.

[0039] Example 9 Take 30 mL of each of the three strains of cyanobacteria cultured in Example 3, centrifuge at 4000 r / min for 2 min, collect the cells, and place them in BG11-N 0 P 3 The culture was cultured in the culture medium for 48 hours, and a sealing film was added to the bottle mouth. The bottle was then placed on a constant temperature shaker at 120 rpm, 1000 Lux, and 25°C for shaking culture. Samples were taken at the 49th hour.

[0040] Example 10 The three cyanobacteria cultured for 24 hours in Example 3 were placed on a constant temperature shaker for shaking culture for 48 hours at 120 rpm, 1000 Lux, and 25° C., and samples were taken at 49 hours.

[0041] In the present invention, the BG11-N 0 P 2 and BG11-N 0 P 3 The formula of culture medium is the same as that of BG11-N 0 The only difference is BG11-N 0 P 2 and BG11-N 0P 3 The concentrations of potassium dihydrogen phosphate are 80 mg / L and 120 mg / L respectively.

[0042] Add Alcian Blue to the three cyanobacteria obtained in the above example, remove the supernatant, and suspend the mycelium in a heterocyst staining buffer. The Alcian Blue preparation method is to weigh 50 mg of the drug and dissolve it in 10 mL of 50% ethanol solution. The heterocyst staining buffer preparation method comprises the following steps: A. Dissolve 0.6055 g of tris(hydroxymethyl)aminomethane in 800 mL of water, add concentrated hydrochloric acid to adjust the pH to 7.8, and then add water to make up to 1 L. After aliquoting, sterilize by high pressure to obtain 5 mM Tris-HCl.

[0043] B. Put 1.4612g of EDTA into Tris-HCl and place in an ultrasonic instrument with an ultrasonic power of 99% and heat to 35°C until it is completely dissolved.

[0044] C. Add 1.7g of BG11 culture medium into the solution to obtain the heterocyst staining buffer.

[0045] After observing the sample suspended in the staining buffer under a microscope, place it in the high-content cell imaging analysis system and follow the steps below: Use a pipette to measure 100 µL of sample and place it in a 96-well plate with a glass bottom. Place the plate into a high-content cell imaging analysis system to overlay the labeled heteromorphic cells, and analyze and quantify the number of heteromorphic cells, cell size, and relative fluorescence intensity.

[0046] The numbers of heterocysts differentiated and formed by the three algae obtained in Examples 1, 3, 4 and 5 under different culture conditions are shown in Table 5. 0 (sampled after Example 3), BG11-N 0 P 2 (sampled after Example 4) and BG11-N 0 P 3 (Sampling after Example 5).

[0047] Table 5 The numbers of heterocysts differentiated from the three algae obtained in Examples 3, 6 and 7 under different culture conditions are shown in Table 6 below. 0 (Sampling after Example 3) BG11-N 0 P 2 (sampled after Example 6) and BG11-N0 P 3 (Sampling after Example 7) Table 6 The numbers of heterocysts differentiated from the three algae obtained in Example 10, Example 8 and Example 9 under different culture conditions are shown in Table 7 below.

[0048] Table 7 From Table 5 (the number of heterocysts differentiated and formed by the three algae obtained in Examples 1, 3, 4 and 5 under different culture conditions) and Figure 2 (Cell size and relative fluorescence intensity) It can be seen that the three algae are respectively in BG11-N 0 、BG11-N 0 P 2 and BG11-N 0 P 3 Induction in culture medium for 24 h, in BG11-N 0 The number of heteromorphic cells induced by BG11-N was the largest. 0 P 2 Next is BG11-N 0 P 3 Among them, BG11-N 0 After induction, the number of heteromorphic cells differentiated by Anabaena was 127% more than that of Nostoc and 911% more than that of Aphanizomenon; the size of heteromorphic cells differentiated by Anabaena was 2% larger than that of Nostoc and 19% larger than that of Aphanizomenon; the relative fluorescence intensity of heteromorphic cells differentiated by Anabaena was 20% stronger than that of Nostoc and 30% stronger than that of Aphanizomenon.

[0049] From Table 6 (the number of heterocysts differentiated under different culture conditions of the three algae obtained in Examples 3, 6 and 7) and Figure 4 (Cell size and relative fluorescence intensity) It can be seen that the three algae are respectively in BG11-N 0 、BG11-N 0 P 2 and BG11-N 0 P 3 Induced in the medium for 24 h, compared with the traditional induction conditions in Example 3 (BG11-N 0), the number of heteromorphic cells induced by the three algae under Example 7 was the largest, followed by Example 3, and the least by Example 6. In Example 7, the number of heteromorphic cells differentiated by Anabaena, Nostoc and Aphanizomenon was 116%, 5% and 9% more than that under the conditions of Example 3; the size of heteromorphic cells differentiated by Anabaena, Nostoc and Aphanizomenon was 11%, 11% and 13% larger than that under the conditions of Example 3, respectively; the relative fluorescence intensity of heteromorphic cells differentiated by Anabaena, Nostoc and Aphanizomenon was 25%, 15% and 7% stronger than that under the conditions of Example 3.

[0050] As shown in Table 7 and Figure 6 The figures show the number, cell size and relative fluorescence intensity of heterocysts differentiated under different culture conditions of the three algae obtained in Example 10, Example 8 and Example 9. 0 、BG11-N 0 P 2 and BG11-N 0 P 3 Compared with the traditional induction conditions in Example 10 (BG11-N 0 ), the number of heteromorphic cells induced by the three algae under Example 9 was the largest. In Example 9, the number of heteromorphic cells differentiated by Anabaena, Nostoc and Aphanizomenon was 73%, 19% and 67% more than that under the conditions of Example 10; the size of heteromorphic cells differentiated by Anabaena, Nostoc and Aphanizomenon was 121%, 33% and 13% larger than that under the conditions of Example 10, respectively; the relative fluorescence intensity of heteromorphic cells differentiated by Anabaena was 100% stronger than that under the conditions of Example 10, and the relative fluorescence intensity of heteromorphic cells differentiated by Nostoc and Aphanizomenon cultured in Example 10 was 35% and 10% stronger than that under the conditions of Example 9.

[0051] All three algae strains were BG11-N in Example 7. 0 P 3 The number of heteromorphic cells induced in the culture was the largest, and the induction time of Example 7 was shortened by half compared with Example 9. The number of heteromorphic cells differentiated from Anabaena, Nostoc and Aphanizomenon induced in Example 7 was 26%, 25% and 9% more than that in Example 9; the size of heteromorphic cells differentiated from Anabaena, Nostoc and Aphanizomenon induced in Example 7 was 64%, 79% and 169% larger than that in Example 9, and the relative fluorescence intensity of heteromorphic cells differentiated from Anabaena, Nostoc and Aphanizomenon induced in Example 7 was 192%, 257% and 267% stronger than that in Example 9. Therefore, under the condition of 3 times phosphorus supplementation after 12 hours of induction, the number of heteromorphic cells differentiated from the three strains of algae showed the best effect.

[0052] Although the formation of heterocysts is mainly triggered by nitrogen deficiency, the sufficiency of phosphorus is an important guarantee for cell metabolism and energy supply in the growth and development of cyanobacteria. The sufficiency of phosphorus sources will affect the overall growth of cyanobacteria, thereby indirectly affecting the development of heterocysts. In an environment where nitrogen is deficient but phosphorus is relatively sufficient (high phosphorus and low nitrogen), more heterocysts will be induced to differentiate to maintain the nitrogen fixation capacity of the population. Sufficient phosphorus sources can indirectly affect the formation of heterocysts by regulating the growth rate, metabolic state and nitrogen demand of cyanobacteria.

[0053] The three strains of cyanobacteria in the present invention are all cyanobacteria that can differentiate heterocysts to fix nitrogen. The nitrogen fixation efficiency of the cyanobacteria is directly related to the characteristics of heterocysts. The number, size and nitrogenase activity of heterocysts are all direct factors that determine the nitrogen fixation efficiency of cyanobacteria. The conventional induction method is to culture in a culture solution with only nitrogen deficiency, while the phosphorus source is supplemented in the nitrogen-deficient culture solution of the present invention. On the one hand, the phosphorus source required by the cyanobacteria can be supplemented, the metabolism and energy supply of the cells can be promoted, and the cell differentiation can be induced; on the other hand, the phosphorus source supplementation can strengthen the degree of nitrogen deficiency and strengthen the induction environment of heterocysts.

[0054] In summary, the above embodiments mainly describe a preferred method of the present invention, including the basic operating steps, main features and advantages of the present invention. The relevant technical personnel of real-time operation should have a certain understanding that the present invention is not limited by the above embodiments. The above has been described in detail with general descriptions and specific implementation schemes. However, on the basis of the present invention, some modifications or improvements can be made to it, which is obvious to those skilled in the art. Therefore, these modifications or improvements made on the basis of not departing from the spirit of the present invention all belong to the scope of protection claimed by the present invention.

Claims

1. A method for rapidly inducing heterocysts of nitrogen-fixing cyanobacteria, characterized in that: Step 1: Induce nitrogen-fixing cyanobacteria in nitrogen-deficient culture medium for 12 hours under light conditions; Step 2: Take out the nitrogen-fixing cyanobacteria induced in step 1, place it in a nitrogen-deficient and phosphorus-supplemented culture medium and continue inducing it for 12 hours; The concentration ratio of the phosphorus source in the culture solution of step one and step two is 1:(2-3).

2. The method for rapid induction of nitrogen-fixing cyanobacteria heterocysts according to claim 1, characterized in that: The concentration ratio of the phosphorus source in the culture solution of step 1 and step 2 is 1:

3.

3. The method for rapid induction of heterocysts of filamentous nitrogen-fixing cyanobacteria according to claim 1, characterized in that: The nitrogen-deficient culture medium in step 1 lacks nitrogen source in its components compared with the BG11 culture medium.

4. The method for rapid induction of nitrogen-fixing cyanobacteria heterocysts according to claim 1, characterized in that: The phosphorus source in the nitrogen-deficient and phosphorus-supplemented culture solution in step 2 and the nitrogen-deficient culture solution in step 1 is dipotassium hydrogen phosphate.

5. The method for rapid induction of nitrogen-fixing cyanobacteria heterocysts according to claim 4, characterized in that: The concentration of dipotassium hydrogen phosphate in the nitrogen-deficient culture medium in step 1 is 40 mg / L, and the concentration of dipotassium hydrogen phosphate in the nitrogen-deficient and phosphorus-supplemented culture medium in step 2 is 80-120 mg / L.

6. The method for rapid induction of nitrogen-fixing cyanobacteria heterocysts according to claim 1, characterized in that: The induction time of step 1 and step 2 is 12 hours.

7. The method for rapid induction of nitrogen-fixing cyanobacteria heterocysts according to claim 1, characterized in that: The nitrogen-fixing cyanobacteria are Anabaena, Nostoc or Aphanizomenon.

8. The method for rapid induction of nitrogen-fixing cyanobacteria heterocysts according to claim 1, characterized in that: The intensity of the light is 1000 Lux, the induction temperature is 20-25° C., and the shaking culture speed is 120 r / min.

9. A nitrogen-fixing cyanobacterial heterocyst, characterized in that: The nitrogen-fixing cyanobacteria heterocysts are prepared by the nitrogen-fixing cyanobacteria heterocysts rapid induction method according to any one of claims 1 to 8.

10. Use of the nitrogen-fixing cyanobacteria heterocystis as claimed in claim 9 in the preparation of microbial nitrogen fertilizer.

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