A biological combined nitrogen fixation system with high ammonium absorption efficiency

Abstract

This invention discloses a highly efficient biological nitrogen fixation system for ammonium absorption, comprising a rice line overexpressing the OSA1 gene and the Ko424 strain. The synergistic effect of these two components enables efficient absorption of ammonium nitrogen. The OSA1 gene encodes the proton pump in the rice cell membrane; overexpression of OSA1 provides increased proton-driven force, promoting root absorption of ammonium nitrogen. The Ko424 strain is a temperature-dependent ammonia-secreting nitrogen-fixing bacterium that provides ammonium nitrogen for gramineous crops. Simultaneously, OSA1 overexpression increases stomatal aperture, promotes carbon dioxide absorption, and enhances photosynthesis. This not only provides more energy for plant nitrogen metabolism but also provides more nutrients as a carbon source for nitrogen fixation by the nitrogen-fixing bacteria colonizing the rice roots. Therefore, this biological nitrogen fixation system promotes rice growth and increases the contribution rate of biological nitrogen and total yield.

Description

Technical Field

[0001] This invention relates to a highly efficient ammonium-absorbing biological co-fixation system, specifically a biological co-fixation system that increases nitrogen contribution through the interaction between highly efficient ammonium-absorbing rice crops and co-fixing nitrogen bacteria, belonging to the field of agricultural biotechnology. Background Technology

[0002] Nitrogen is one of the major nutrients limiting crop growth and yield in agricultural production. Studies by Zhang et al. have shown that the cell membrane proton pump (PMH)... + -ATPase) is involved in the regulation of ammonium nitrogen uptake in rice. It has been clarified that overexpression of the proton pump gene OSA1 in rice can promote ammonium acquisition and assimilation, increasing photosynthetic efficiency (Zhang, Maoxing, et al., "Plasma membrane H...). + -ATPase overexpression increases rice yield via simultaneous enhancement of nutrient uptake and photosynthesis. (Nature Communications 12(2021):735). Field experiments showed that OSA1 overexpression significantly increased rice yield and nitrogen use efficiency. Tang et al. developed a co-nitrogen-fixing bacterium, Ko424, which, as a temperature-dependent ammonia secretion donor, has been shown to provide ammonium nitrogen to various crops, including rice and corn (Tang, Yuqian, et al., "Diurnal switches in diazotrophic lifestyle increase nitrogen contribution to cereals." Nature Communications 14(2023):7516). However, in natural systems, the nitrogen contribution efficiency of Ko424 is still limited by the ammonium uptake capacity of the host plant, and current technologies have not yet combined the two to form a synergistic system. In addition, traditional fertilization relying on chemical nitrogen fertilizers easily leads to environmental pollution and resource waste. Summary of the Invention

[0003] The purpose of this invention is to provide a highly efficient biological nitrogen fixation system for ammonium absorption, aiming to further improve the biological nitrogen contribution efficiency of the combined nitrogen fixation system, thereby reducing the use of chemical nitrogen fertilizers and increasing crop yield.

[0004] The present invention provides a highly efficient biological nitrogen fixation system for ammonium absorption, comprising a rice line overexpressing the OSA1 gene and the Ko424 strain. The synergistic effect of these two genes enables efficient absorption of ammonium nitrogen. The OSA1 gene encodes a proton pump in the rice cell membrane; its overexpression enhances proton-driven force and promotes ammonium absorption by the rice plant. The Ko424 strain is a temperature-dependent ammonia-secreting nitrogen-fixing bacterium that provides ammonium nitrogen to gramineous crops in the combined nitrogen fixation system.

[0005] The construction of the highly efficient ammonium-absorbing biological combined nitrogen fixation system includes:

[0006] A. Plant chassis construction, implemented as follows:

[0007] Rice materials overexpressing the OSA1 gene (OS) showed significantly higher root ammonium uptake efficiency than wild-type (WT) and knockout lines (NE) that knocked out the OSA1 gene.

[0008] The rice material overexpressing the OSA1 gene can be constructed from wild-type rice (Oryza sativa L.ssp.). In some specific embodiments of the present invention, japonica rice (Nipponbare) is selected to construct the OSA1 gene overexpression line. The specific construction method is as follows: the open reading frame of the OSA1 gene is amplified using gene-specific primers; the fragment is treated with restriction endonuclease and inserted into the transgenic vector CaMV-35S, and its downstream gene is expressed by the CaMV-35S promoter; the sequenced vector is transformed by Agrobacterium-mediated transformation of rice embryo callus, and the resulting homozygous T2 or T3 lines are used for phenotypic analysis.

[0009] B. The construction of microbial components is achieved as follows:

[0010] Reference Nature Communications 14(2023):7516. Using *Klebsiella oxytoca* M5al, a nitrogen-fixing bacterium that colonizes the roots of gramineous crops, as the starting strain, the Ko424 point mutant was constructed using the temperature-sensitive plasmid pKD46 expressing Red recombinase and λRed-mediated homologous recombination. Plasmids or DNA fragments were transformed into wild-type strains via electroporation or by binding to ST18 cells. Initially, a glnA-deficient strain with an antibiotic resistance cassette was obtained via electroporation. Then, a glnA homologous fragment containing a single-point mutation (P95L) was transferred into the glnA::Km knockout mutant. The Ko424 mutant—a mutant strain carrying glutamine synthase (GS, glnA)—was obtained in a restrictive medium containing ammonium (but no glutamine). This strain secretes ammonium nitrogen in a temperature-dependent manner, providing biological nitrogen for gramineous plants.

[0011] The above-mentioned highly efficient ammonium-absorbing biological nitrogen fixation system promotes the absorption of ammonium nitrogen nutrients in rice, increases crop yield, and reduces the use of chemical nitrogen fertilizers. The operation process is as follows:

[0012] 1) Pretreatment of the plants to be inoculated, including:

[0013] 1a. After disinfecting the surface of the OSA1 gene overexpression line rice seeds, soak them in a saturated CaSO4 solution overnight, and then wash them with sterile water;

[0014] 1b. Rice seeds germinate in hydroponic solution for approximately 7 days;

[0015] 1c. Select seedlings with intact root systems and good growth for bacterial inoculation experiments;

[0016] 2) Preparation of nitrogen-fixing bacterial solution, including:

[0017] 2a. The Ko424 strain was continuously streaked on culture plates for subculturing to activate the strain;

[0018] 2b. Inoculate the activated Ko424 strain into liquid culture medium and culture until the logarithmic growth phase. Wash with a nutrient-free medium (mainly carbon and nitrogen sources), centrifuge, resuspend and concentrate, and adjust the cell count to 102. 7 ~10 9 cells / mL;

[0019] 3) Inoculation with nitrogen-fixing bacteria, including:

[0020] 3a. Under sterile conditions, remove the entire embryo from the seedlings selected in step 1) and place them in a hydroponic nutrient solution such as Kimura B nutrient solution for later use.

[0021] 3b. Immerse the entire root system of the seedling in the bacterial solution prepared in step 2) and inoculate with nitrogen-fixing bacteria, and keep for 0.5–1.5 hours;

[0022] 4. Seedlings inoculated with nitrogen-fixing bacteria are subjected to combined nitrogen-fixing culture. In practice, seedlings can be directly transplanted to different culture environments, such as hydroponic systems and soil culture systems.

[0023] In some embodiments of the present invention, seedlings inoculated with nitrogen-fixing bacteria are transplanted into Kimura B nutrient solution for combined nitrogen-fixing culture. The entire hydroponic system is placed in a light incubator and cultured under a cyclical change of daytime (high temperature) and nighttime (low temperature). The aerated parts of the hydroponic system are wrapped with a sealing film to minimize close contact between the plant roots and fresh air; and a certain volume of [unspecified substance] is added. 15The efficiency of nitrogen fixation in the system was reflected by subsequent isotope mass spectrometry analysis of N2 gas. During cultivation, a small amount of distilled water and nutrient solution could be added to prevent rapid evaporation of water from the hydroponic system.

[0024] The method for reflecting the combined nitrogen fixation efficiency of the system through isotope mass spectrometry detection is as follows: the entire combined nitrogen fixation system is placed in a sealed plastic bag for cultivation, and a certain volume of gas is added through gas replacement inside the bag. 15 N2 gas was used to cultivate the plants under the same light and temperature conditions, and then the plants were harvested to detect and calculate the bio-nitrogen incorporation efficiency provided to the host plants by the nitrogen-fixing bacteria.

[0025] The beneficial effects of this invention are:

[0026] This invention provides a biological combined nitrogen fixation system that can efficiently absorb ammonium. In this system, overexpression of OSA1 provides more proton-driven force, promoting root uptake of ammonium nitrogen, and through the H2 produced by ammonium assimilation... + Timely expulsion of ammonium nitrogen from the cell membrane promotes its assimilation and ensures the absorption of ammonium secreted by bacteria by rice. Simultaneously, overexpression of OSA1 increases stomatal aperture, promoting carbon dioxide absorption. This enhanced leaf photosynthesis not only provides more energy for plant nitrogen metabolism but also provides more nutrients as a carbon source for nitrogen-fixing bacteria colonizing the rice roots. Therefore, this combined nitrogen-fixing system further promotes rice growth, increases the contribution of biological nitrogen, and improves overall yield. Attached Figure Description

[0027] Figure 1 This is the result of the rice methylammonium toxicity experiment in Example 1. Kimura B nutrient solution was added to two hydroponic boxes. The hydroponic box on the left was the control (CK), and the hydroponic box on the right had an additional 10 mM methylammonium (MeA) added to the Kimura B nutrient solution as an ammonium analog. Wild-type japonica rice (WT), OSA1 gene knockout (NE), and OSA1 gene overexpression line (OS) rice were planted in each hydroponic box from left to right.

[0028] Figure 2 This is the result of Example 2 regarding the detection efficiency of biological nitrogen incorporation in the combined nitrogen fixation system. A. From left to right, the results show the plant growth of rice with the OSA1 gene knocked out (NE), wild-type japonica rice (WT), and OSA1 gene overexpression line (OS) after inoculation with Ko424 and cultured in sealed plastic bags for 8 days; B. Showing the amount of pheophytin in young rice leaves after Ko424 inoculation. 15 Differences in nitrogen isotope abundance. Detailed Implementation

[0029] The technical solution and its effects of the present invention will be further illustrated below with reference to the accompanying drawings and embodiments. The described embodiments are exemplary and do not imply any limitation on the present invention in any way.

[0030] Example 1: Rice Methylammonium Toxicity Experiment

[0031] Ammonium ion uptake by plant roots occurs through specific ammonium transporters (AMTs). The ammonium uptake efficiency of different plant materials can be observed by adding methylammonium (MeA, which has certain toxic effects) to the plant culture medium. Rice seeds were sterilized and germinated in sterile water. Seedlings with uniform growth were selected and transferred to Kimura B nutrient solution with or without methylammonium (concentrations of 5 mM, 10 mM, or 15 mM). Wild-type japonica rice (WT), OSA1 gene knockout (NE), and OSA1 gene overexpression line (OS) were cultured for 14 days at 30℃ under light / dark (12h / 12h) conditions, and growth differences among the different plant materials were observed. As an ammonium analog, methylammonium uptake can cause plant toxicity; at the same dosage, materials with high uptake showed partial growth inhibition. Results showed that the OSA1 gene overexpression line (OS) had significantly higher ammonium analog uptake than wild japonica rice and the knockout line. Figure 1 This result indicates that OS rice possesses physiological characteristics for efficient ammonium absorption.

[0032] Example 2: Experimental Assessment of the Biological Nitrogen Contribution of a High-Efficiency Ammonium Absorption Combined with Nitrogen Fixation System

[0033] In the inoculation experiment of the nitrogen-fixing and ammonium-secreting bacteria Ko424, surface-sterilized rice seeds were washed 4-5 times with sterile distilled water, then soaked overnight in saturated CaSO4, washed 6-7 times with sterile distilled water, and transferred to sterile water to await germination. About a week later, rice seedlings from different backgrounds with completely uniform growth were selected. To limit the transfer of stored nutrients (especially nitrogen sources), the entire embryo at the root of the seedling was removed, and the entire root system was then soaked in Ko424 bacterial solution for 1 hour. The bacterial solution required prior cultivation of Ko424 to OD in LB medium. 600 The concentration was 0.6-0.8. Subsequently, bacterial cells were collected by centrifugation, washed, and resuspended to the desired cell density (10⁻⁶). 9 / mL). After inoculation, the seedlings were transferred to glass test tubes, and thin sponges were wrapped around the junction of the roots and stems to provide support. Kimura B nutrient solution was added to the test tubes, and the entire hydroponic system was placed in a transparent, airtight plastic bag, with 50% of the gas space being... 15Nitrogen gas was used for replacement, resulting in a final oxygen concentration of 20% in the gas mixture. 1% CO2 was added daily to maintain the yellow color of the CO2 indicator in the bag. Eight days after planting, chlorophyll was extracted from the third young leaf and converted to pheophytin using concentrated hydrochloric acid. The pheophytin was analyzed using an LTQ Orbitrap XL mass spectrometry system (Orbitrap XL, Thermo Fisher) in electrospray ionization and positive ion mode. 15 Nitrogen isotope abundance. Standards (chlorophyll a, Sigma-Aldrich) were prepared using the same chlorophyll conversion method to calculate nitrogen abundance in the samples. 15 Nitrogen isotope abundance. Plant growth status as follows: Figure 2 As shown in Figure A, the results indicated that the overexpression line (OS) exhibited a significant increase in plant height after inoculation with the Ko424 ammonium-secreting strain, compared to the wild-type (WT) and knockout (NE) lines. Phthaphylline was further extracted from the young leaves, and its concentration in the samples was detected and calculated using an LTQ Orbitrap XL mass spectrometry system. 15 Nitrogen isotope doping abundance, results are as follows Figure 2 As shown in Figure B, the overexpression line (OS) has a significantly increased contribution of biological nitrogen from Ko424.

[0034] Finally, it should be noted that the purpose of disclosing the embodiments is to help further understand the present invention. Those skilled in the art should understand that various substitutions and modifications are possible without departing from the spirit and scope of the present invention and the appended claims. Therefore, the present invention should not be limited to the content disclosed in the embodiments, and the scope of protection of the present invention is defined by the claims.

Claims

1. A highly efficient biological nitrogen fixation system for ammonium uptake, comprising a rice line overexpressing the OSA1 gene and the Ko424 strain, wherein, The OSA1 gene encodes a proton pump in rice cell membranes, and the Ko424 strain is a temperature-dependent ammonia-secreting nitrogen-fixing bacterium. The two work synergistically to achieve efficient absorption of ammonium nitrogen nutrition.

2. The biological combined nitrogen fixation system as described in claim 1, characterized in that, The Ko424 strain was colonized in the roots of rice plants that overexpressed the OSA1 gene.

3. A method for constructing the biological combined nitrogen fixation system according to claim 1, comprising: A. Constructing rice lines overexpressing the OSA1 gene; B. Using Klebsiella oxytoca M5al as the starting strain, the glutamine synthase gene glnA was mutated to obtain the Ko424 strain.

4. The construction method as described in claim 3, characterized in that, The rice lines overexpressing the OSA1 gene were constructed from wild-type japonica rice.

5. The construction method as described in claim 3, characterized in that, Step A first constructs an expression vector that expresses the OSA1 gene driven by the CaMV-35S promoter, and then transforms rice with it to obtain rice lines that overexpress the OSA1 gene.

6. The construction method as described in claim 3, characterized in that, The glnA mutation mentioned in step B is a P95L single-point mutation.

7. The application of the biological combined nitrogen fixation system of claim 1 in rice production.

8. The application as described in claim 7, characterized in that, Promoting the absorption of ammonium nitrogen in rice using the biological combined nitrogen fixation system according to claim 1 includes the following steps: 1) Pretreatment of plants to be inoculated: After disinfecting the surface of rice seeds overexpressing the OSA1 gene, soak them in a saturated CaSO4 solution overnight, and then wash them with sterile water; then germinate the seeds in hydroponic solution, and select seedlings with intact roots and good growth for bacterial inoculation. 2) Preparation of nitrogen-fixing bacterial suspension: Ko424 strain was activated by continuous streaking on agar plates, then inoculated into liquid culture medium and cultured to the logarithmic growth phase. The cells were washed with a medium free of carbon and nitrogen sources, centrifuged, resuspended, and concentrated. The cell count was adjusted to 102. 7 ~10 9 cells / mL; 3) Inoculation with nitrogen-fixing bacteria: Under sterile conditions, remove the entire embryo part from the seedlings selected in step 1), and then immerse the entire root system of the seedlings in the bacterial solution prepared in step 2) to inoculate with nitrogen-fixing bacteria. 4) Combine nitrogen fixation culture with seedlings inoculated with nitrogen-fixing bacteria.

9. The application as described in claim 8, characterized in that, In step 1), the seeds are germinated and cultured in hydroponic solution for 7 days.

10. The application as described in claim 8, characterized in that, In step 3), the seedling roots are soaked in nitrogen-fixing bacteria solution for 0.5 to 1.5 hours.

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