Application of transcription factor sty2 in regulating nodule development

Abstract

The application provides application of transcription factor STY2 in regulation of nodule development. The application provides a method for improving nitrogen fixation capacity of a nodule plant or a method for preparing a nodule plant with increased nitrogen fixation capacity, comprising: up-regulating expression or activity of STY2 in the nodule plant.

Description

Technical Field

[0001] This invention belongs to the field of botany, and more specifically, this invention relates to the application of transcription factor STY2 in regulating root nodule development. Background Technology

[0002] Nitrogen is a macronutrient essential for plant growth and development, and also one of the main limiting factors for crop yield. The Earth is rich in nitrogen, with over 80% of the atmosphere being nitrogen gas. However, it cannot be directly absorbed and utilized by plants; only by fixing atmospheric nitrogen and reducing it to ammonia can it be directly used by plants. Nitrogen fixation includes three methods: chemical nitrogen fixation, biological nitrogen fixation, and physical nitrogen fixation. While industrial chemical nitrogen fixation produces fertilizers that meet the nitrogen requirements of crops, it also leads to a series of energy and environmental problems, such as the consumption of large amounts of fossil fuels, greenhouse gas emissions, and eutrophication of water bodies. Therefore, seeking environmentally friendly and efficient nitrogen fixation methods has become an urgent need for sustainable agricultural development.

[0003] In nature, nitrogenases in some nitrogen-fixing microorganisms can convert nitrogen gas into ammonia, which plants can directly absorb and utilize—this is known as biological nitrogen fixation. Biological nitrogen fixation provides approximately three-quarters of the global nitrogen required by plants in Earth's ecosystems. Therefore, biological nitrogen fixation is the largest "natural nitrogen fertilizer factory" on Earth. Biological nitrogen fixation is divided into three different systems: autotrophic nitrogen fixation, associative nitrogen fixation, and symbiotic nitrogen fixation. Among them, the symbiotic nitrogen fixation system is the most important nitrogen fixation system in nature, especially the symbiotic nitrogen fixation between leguminous plants and rhizobia, which has the highest nitrogen fixation efficiency, accounting for about 60% of nitrogen fixation in terrestrial ecosystems. Statistics show that leguminous plants and rhizobia fix 4 × 10⁻⁶ nitrogen through symbiotic nitrogen fixation. 7 –5×10 7 The amount of nitrogen available to plants per ton per year is the most important source of nitrogen fertilizer for sustainable agriculture. Therefore, elucidating the symbiotic nitrogen fixation mechanism between legumes and rhizobia, and making full use of the symbiotic nitrogen fixation capacity of legumes, has significant practical application value for "reducing fertilizer use and increasing efficiency" in agricultural production.

[0004] Rhizobia can induce the formation of new lateral organs—nodules—in the roots of leguminous plants. Nodule formation involves the reactivation of epidermal and cortical cells. Rhizobia induce root hairs to bend and deform, causing the root hair cell membranes to invaginate and form a tubular structure—the infection thread. Rhizobia extend along the infection thread to the cortical cells. Simultaneously, cortical cells are activated, undergoing cell division to form nodule primordia. After differentiation, rhizobia are released from the infection thread into the nodule primordia, where they are encapsulated by the plant body membrane and differentiate into nitrogen-fixing bacteroids. Rhizobial infection and nodule organ development must be coordinated to form nitrogen-fixing nodules.

[0005] The most important property of root nodules is their ability to accommodate rhizobia for symbiotic nitrogen fixation. However, the regulatory mechanisms by which plant cells accommodate rhizobia colonization remain unclear. Interestingly, rhizobia cannot invade dividing root nodule primordia; they can only be infected and colonized after the cells have differentiated. Therefore, root nodule cell differentiation is a prerequisite for the establishment of symbiosis.

[0006] Therefore, there is an urgent need in this field to conduct in-depth research on the genetic basis of root nodule cell differentiation in legumes and to elucidate the molecular mechanism of root nodule symbiosis, so as to improve the nitrogen fixation efficiency of legumes themselves and lay a theoretical foundation for symbiotic nitrogen fixation in non-legume plants. Summary of the Invention

[0007] The purpose of this invention is to provide the application of transcription factor STY2 in regulating root nodule development.

[0008] In a first aspect of the invention, a method for improving the nitrogen fixation capacity of rhizobia or a method for preparing rhizobia with increased nitrogen fixation capacity is provided, comprising: upregulating the expression or activity of STY2 in rhizobia.

[0009] In one or more embodiments, upregulating the expression or activity of STY2 in plants includes:

[0010] Introduce the STY2 coding gene or an expression construct or vector containing the coding gene into plants;

[0011] Promote STY2 expression by expressing an enhancing promoter or a tissue-specific promoter; or,

[0012] Enhancers promote STY2 expression.

[0013] In one or more embodiments, the expression or activity of STY2 in rhizobia is upregulated in rhizobia with low or absent STY2 expression.

[0014] In a second aspect of the invention, the use of a STY2 upregulator is provided for improving the nitrogen fixation capacity of rhizobium plants.

[0015] In one or more embodiments, the upregulator includes: an exogenous STY2 encoding gene or an expression construct or vector containing the encoding gene.

[0016] In one or more embodiments, the expression construct includes an enhanced promoter, a tissue-specific promoter, or an enhancer.

[0017] In one or more embodiments, the improvement of nitrogen fixation capacity of rhizobium plants includes: regulating the formation of rhizobium-infecting cells, promoting rhizobium development, promoting the development and maturation of red rhizobiums, improving the nitrogen fixation capacity of rhizobium plant roots, promoting nitrogen utilization by rhizobium plants, reducing the nitrogen fertilizer requirement of rhizobium plants, increasing rhizobium plant biomass, or increasing rhizobium plant yield.

[0018] In one or more embodiments, the root nodule plant includes or is selected from plants of the group consisting of: plants expressing the STY2 gene or protein, root nodule plants, non-leguminous plants, grasses, and cruciferous plants.

[0019] In one or more embodiments, the root nodule plant is a legume.

[0020] In one or more embodiments, the legumes include: Tribulus terrestris, alfalfa, tribulus terrestris alfalfa, soybean, broad bean, pea, mung bean, red bean, cowpea, common bean, hyacinth bean, pigeon pea, peanut, milkvetch, broad bean, sagebrush, albizia, rosewood, soapberry, ebony, red bean, pagoda tree, horsethorn, pagoda tree flower, indigo, sappanwood, gum, gum arabic, astragalus gum, copal gum, Indian hemp, kudzu, and spider vine.

[0021] In one or more embodiments, the amino acid sequence of the STY2 protein is selected from the group consisting of:

[0022] (i) A protein having the amino acid sequence shown in SEQ ID NO:2;

[0023] (ii) A protein derived from (i) having the regulatory trait function formed by substituting, deleting or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID NO:2;

[0024] (iii) A protein whose amino acid sequence has ≥80% homology with the amino acid sequence shown in SEQ ID NO:2 and has the function of regulating the aforementioned traits;

[0025] (iv) The active fragment of the polypeptide with the amino acid sequence shown in SEQ ID NO:2; or,

[0026] (v) A protein formed by adding a tag sequence or restriction enzyme site sequence to the N or C end of a polypeptide with the amino acid sequence shown in SEQ ID NO:2, or by adding a signal peptide sequence to its N end.

[0027] In one or more embodiments, the amino acid sequence of the STY2 protein is shown in SEQ ID NO:2.

[0028] In a third aspect of the invention, there is a use of the root nodule plant STY2 as a molecular marker for identifying root nodule plant traits, or as a molecular marker for targeted screening of plants; wherein the trait includes: nitrogen fixation capacity of the root nodule plant.

[0029] In one or more embodiments, the nitrogen fixation capacity of the rhizobium includes: formation of nodule-infecting cells, nodule development, red nodule development and maturation, nitrogen fixation capacity of the roots of the rhizobium, nitrogen utilization rate of the rhizobium, nitrogen fertilizer requirement of the rhizobium, biomass of the rhizobium, or yield of the rhizobium.

[0030] In a fourth aspect of the invention, a method for selecting or identifying root-nodule plants is provided, the method comprising: identifying the expression of STY2 protein or its gene in a test plant; if the STY2 protein or its gene in the test plant is highly expressed or highly active, then the plant is a plant with high nitrogen fixation capacity; if the STY2 protein or its gene in the test plant is low expressed or low active, then the plant is a plant with low nitrogen fixation capacity.

[0031] In one or more embodiments, the nitrogen fixation capacity of the rhizobium includes: formation of nodule-infecting cells, nodule development, red nodule development and maturation, nitrogen fixation capacity of the roots of the rhizobium, nitrogen utilization rate of the rhizobium, nitrogen fertilizer requirement of the rhizobium, biomass of the rhizobium, or yield of the rhizobium.

[0032] In a fifth aspect of the invention, a method is provided for screening substances (including potential substances) that regulate the nitrogen fixation capacity of rhizobium plants, comprising:

[0033] (1) Add the candidate substance to the system expressing STY2;

[0034] (2) Detect the system and observe the expression or activity of STY2. If the expression or activity of STY2 is increased, it indicates that the candidate substance is a substance that can be used to increase the nitrogen fixation capacity of rhizobium plants; if the expression or activity of STY2 is decreased, it indicates that the candidate substance is a substance that can be used to reduce the nitrogen fixation capacity of rhizobium plants.

[0035] Other aspects of the invention will be apparent to those skilled in the art from the disclosure herein. Attached Figure Description

[0036] Figure 1 Expression patterns and subcellular localization of the MtSTY2 gene.

[0037] (A) Heatmap of expression of MtSTY2 and root nodule development-related genes in wild type (WT) and spontaneous nodule mutant (spd1).

[0038] (B) p35S-GFP-MtSTY2 coincides with the nuclear localization signal of the nuclear marker gene NLS-DsRed, indicating that MtSTY2 is expressed in the cell nucleus, with the scale line at 20 μm.

[0039] (C) Expression levels of MtSTY2 relative to the internal reference gene MtUbiquitin in roots and root nodules.

[0040] (D) The tissue expression pattern of MtSTY2 driven by the MtSTY2 promoter is shown in the results, indicating that MtSTY2 is expressed in the root nodule primordium and the apical meristem of the root nodule. M:meristem. The white scale bar is 1 mm, and the black scale bar is 100 μm.

[0041] Figure 2 Identification of Mtsty2 mutant.

[0042] (A) Schematic diagram of the insertion site of transposon Tnt1 into the MtSTY2 genome in the MtSTY2 mutant. The black solid lines represent introns and the 5' / 3'-UTR transcriptional untranslated region, the black squares represent exons, the triangle above the MtSTY2 gene indicates the Tnt1 insertion site, and the arrows indicate the locations of primers for mutant identification.

[0043] (B) PCR identification of the Mtsty2 homozygous mutant.

[0044] (C) Expression levels of MtSTY2 relative to the internal reference gene MtUbiquitin in WT and mutant roots. Values ​​are mean ± SD. An asterisk indicates a significant difference compared to wild type (t-test, ***P<0.001).

[0045] Figure 3 Phenotypic analysis of the Mtsty2 mutant.

[0046] (A) Representative images of wild-type and Mtsty2 plants 3 weeks after inoculation with S. meliloti 1021 rhizobium. Mtsty2 leaves turned yellow, showing a clear nitrogen deficiency phenotype, with the scale line at 1 cm.

[0047] (B) Wild type and Mtsty2 nodulation phenotype. Mtsty2 forms white root nodules, indicating that the root nodules cannot fix nitrogen normally, with the scale line at 1 mm.

[0048] (C) Statistical analysis of the number of root nodules after 1-3 wpi of rhizobium inoculation. An asterisk indicates a significant difference compared to the wild type (***P<0.001, ns, no significant, by t-test).

[0049] Figure 4 Wild type, Mtsty2 mutant root nodule development.

[0050] (A) Results of semi-thin sections of root nodules from WT and Mtsty2.

[0051] (B) Statistical analysis of the number of infected cells in wild-type and Mtsty2 root nodules. Sections of Mtsty2 mutant and wild-type root nodules were prepared, and the number of infected cells in the root nodules was counted using ImageJ.

[0052] (C) Nitrogenase activity assay in root nodules of Mtsty2 mutant and wild type.

[0053] (D) Statistical analysis of infection lines in root nodules of the Mtsty2 mutant and wild type. An asterisk indicates a significant difference compared to the wild type (**P<0.01, ***P<0.001, by t-test). Scale bar is 100 μm.

[0054] Figure 5 Genetic complementation of the Mtsty2 mutant.

[0055] (A) Root nodule phenotype of WT and Mtsty2 transformed with empty vector (EV) or pMtSTY2:STY2. Compared with the empty vector, the roots of the pMtSTY2:STY2 transgenic plants can form red nodules, indicating that MtSTY2 can fill in mutants.

[0056] (B) Statistical analysis of the number of root nodules in replanted plants. Different letters indicate statistically significant differences (P<0.05, one-way ANOVA). Scale bar is 1 mm. Detailed Implementation

[0057] Legume-rhizobium symbiotic nitrogen fixation is the most important source of nitrogen fertilizer for sustainable agriculture. Studying the mechanisms of symbiotic nitrogen fixation helps address two key issues: firstly, it can improve the nitrogen fixation efficiency of legumes themselves; secondly, it provides a scientific basis for exploring the potential of symbiotic nitrogen fixation in non-legume plants. Currently, our understanding of the molecular mechanisms of symbiotic nitrogen fixation in legumes is still limited, such as how rhizobium stem cells differentiate into nitrogen-fixing cells capable of accommodating rhizobium colonization.

[0058] Through in-depth research, the inventors have for the first time revealed that STY2 is a transcription factor highly expressed in alfalfa root nodules, and it is crucial for nodule development, especially the development of nitrogen-fixing red nodules. Further research shows that STY2 regulates the formation of nodule-infecting cells, and that promoting STY2 gene expression via a promoter can regenerate the mature red nodule phenotype that the sty2 mutant cannot form. Therefore, this gene can be used for plant improvement, enhancing the symbiotic nitrogen fixation efficiency of leguminous crops, and providing a theoretical basis and guidance for exploring symbiotic nitrogen fixation in non-leguminous crops.

[0059] Genes, proteins and plants

[0060] As used in this article, the term "STY2" refers to a gene or protein in rhizobium plants that is homologous to the STY2 gene or protein derived from alfalfa, contains substantially the same structural domains, and has substantially the same function.

[0061] In this invention, the STY2 protein also includes fragments, derivatives, and analogs thereof. As used herein, the terms "fragment," "derivative," and "analyte" refer to protein fragments that substantially retain the same biological function or activity as the polypeptide, and may be (i) proteins with one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) substituted, where such substituted amino acid residues may or may not be encoded by the genetic code; or (ii) proteins having substituent groups in one or more amino acid residues; or (iii) proteins formed by the fusion of additional amino acid sequences into the protein sequence, etc. These fragments, derivatives, and analogs are well known to those skilled in the art as defined herein. All bioactive fragments of the STY2 polypeptide can be used in this invention.

[0062] In this invention, the term "STY2 protein" refers to a protein having the SEQ ID NO:2 sequence with STY2 protein activity. This term also includes variations of the SEQ ID NO:2 sequence having the same function as the STY2 protein. These variations include (but are not limited to): deletions, insertions, and / or substitutions of several amino acids (typically 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10, and even more preferably 1-8 or 1-5); and the addition or deletion of one or more amino acids (typically up to 20, preferably up to 10, and more preferably up to 5) at the C-terminus and / or N-terminus. For example, in the art, substitution with amino acids of similar or comparable properties generally does not alter the function of the protein. Similarly, the addition or deletion of one or more amino acids at the C-terminus and / or N-terminus generally does not alter the function of the protein.

[0063] The present invention also includes a polynucleotide (gene) encoding the polypeptide, such as a polynucleotide of the nucleotide sequence shown in SEQ ID NO:1 or a degenerate sequence thereof, which may encode the STY2 protein of SEQ ID NO:2; or a polynucleotide of the nucleotide sequence shown in SEQ ID NO:2 or a degenerate sequence thereof, which may encode the STY2 protein of SEQ ID NO:2. The term "encoding gene" may include a polynucleotide encoding the protein, or it may include a polynucleotide that also includes additional coding and / or non-coding sequences.

[0064] It should be understood that although the STY2 gene of the present invention is preferably obtained from legumes, particularly alfalfa, other genes obtained from other plants that are highly homologous to the alfalfa STY2 gene (e.g., having more than 80%, such as 85%, 90%, 95%, or even 98% sequence identity) or that are degenerate with the said gene are also within the scope of the present invention. Methods and tools for comparing sequence identity are also well known in the art, such as BLAST.

[0065] Vectors containing the said coding sequence, and host cells genetically engineered using the said vector or polypeptide coding sequence, are also included in this invention. Methods well known to those skilled in the art can be used to construct suitable expression vectors.

[0066] The host cell is usually a plant cell. Transformation of plants can generally be achieved using methods such as Agrobacterium-mediated transformation or gene gun transformation, for example, the leaf disc method or rice embryo transformation; Agrobacterium-mediated transformation is preferred. Transformed plant cells, tissues, or organs can be regenerated into plants using conventional methods, thereby obtaining plants with altered traits compared to the wild type.

[0067] As used herein, the plants referred to include, but are not limited to, plants selected from the following group: plants expressing the STY2 gene or protein; root nodules; non-leguminous plants; grasses and / or cruciferous plants.

[0068] As used herein, "nodule-forming plants" primarily refers to terrestrial crops with edible tubers or rhizomes. This includes, but is not limited to: tuberous plants of the Euphorbiaceae family such as alfalfa (including, for example, clover), tuberous plants of the Convolvulaceae family such as sweet potato, tuberous plants of the Solanaceae family such as potato, tuberous plants of the Dioscoreaceae family such as yam, tuberous plants of the Araceae family such as taro and konjac, tuberous plants of the Fabaceae family such as kudzu root, and tuberous plants of the Asteraceae family such as Jerusalem artichoke and yacon. Preferably, the "nodule-forming plants" referred to are "Fabaceae plants".

[0069] The root nodule plants preferably include legumes; more preferably, they include (but are not limited to): edible plants such as soybeans, broad beans, peas, mung beans, red beans, cowpeas, kidney beans, hyacinth beans, pigeon peas, peanuts, etc.; fodder plants such as tribulus terrestris, alfalfa, tribulus terrestris and alfalfa, milkvetch, broad beans, and sagebrush, etc.; timber plants such as mimosa, rosewood, soapberry, ebony, red bean, and locust, etc.; dye plants such as horse broom, locust flower, indigo, and sappanwood, etc.; gums, etc.; resins such as gum arabic, astragalus gum, and copal gum, etc.; fiber plants such as hemp and kudzu, etc.; and oilseed plants such as soybeans and peanuts, etc.

[0070] It should be understood that, given the guidance of the technical solutions of this invention, those skilled in the art will readily conceive of using various legume crops to achieve the same or similar technical effects, and these variations are also included in this invention.

[0071] As used herein, “non-leguminous plants” preferably include grasses and / or cruciferous plants.

[0072] The grasses mentioned herein preferably include (but are not limited to): rice, barley, wheat, oats, rye, corn, and sorghum.

[0073] The cruciferous plants mentioned herein preferably include (but are not limited to): watercress, rapeseed, broccoli, cabbage, white clover, wild wood ear fungus, Chinese cabbage, English cabbage, quince, and taro.

[0074] Regarding "control plants," selecting appropriate control plants is a routine part of experimental design. These can include corresponding wild-type plants or transgenic plants without the target gene. Control plants are generally the same plant species or even varieties of the same species or class as the plant being evaluated. Control plants can also be individuals from transgenic plants that have lost their transgenic components due to segregation. As used in this article, control plants refer not only to whole plants but also to plant parts, including seeds and seed portions.

[0075] Applications or methods to improve nitrogen fixation capacity in rhizobium plants

[0076] Based on the inventors' new discovery, this invention provides the use of the STY2 protein or its encoding gene for enhancing the nitrogen fixation capacity of rhizotrophic plants. This invention also provides a method for enhancing the nitrogen fixation capacity of rhizotrophic plants or a method for preparing rhizotrophic plants with increased nitrogen fixation capacity, the method comprising: increasing the expression or activity of STY2 in the rhizotrophic plants.

[0077] In this article, "improving the nitrogen fixation capacity of root nodules" includes, but is not limited to: regulating the formation of root nodule-infecting cells, promoting root nodule development, promoting the development and maturation of red root nodules, improving the nitrogen fixation capacity of root nodules, promoting the utilization of nitrogen by root nodules, reducing the demand for nitrogen fertilizer by root nodules, increasing the biomass of root nodules, or increasing the yield of root nodules.

[0078] In this article, the terms "upregulation," "enhancement," and "promotion" include "upregulation" and "promotion" of protein activity or "upregulation," "enhancement," and "promotion" of protein expression. The "upregulation," "enhancement," or "promotion" is typically significant, such as an upregulation, enhancement, or promotion of 30%, 40%, 50%, 60%, or higher.

[0079] Any substance that can enhance the activity of the STY2 protein, improve the stability of the STY2 gene or its encoded protein, upregulate the expression of the STY2 gene, or increase the effective duration of the STY2 protein can be used in this invention as an effective substance for improving the nitrogen fixation capacity of plants. These substances can be compounds, small chemical molecules, or biomolecules. The biomolecules can be at the nucleic acid level (including DNA and RNA) or at the protein level.

[0080] This invention also relates to upregulators of the STY2 protein or its encoding gene and their uses. Since upregulators of STY2 can regulate STY2 expression and / or activity, these upregulators can also enhance nitrogen fixation capacity in plants by affecting STY2, thereby improving plant growth.

[0081] Once the uses of the STY2 protein are understood, various methods well-known to those skilled in the art can be used to upregulate its expression. For example, an expression unit carrying the STY2 coding gene (such as an expression vector or virus) can be delivered to the target site via a certain pathway, thereby enabling the expression of the active STY2 protein.

[0082] In one embodiment of the present invention, the coding gene for the STY2 protein is cloned into a suitable vector, and the recombinant vector carrying the foreign gene is introduced into plant cells capable of expressing the STY2 protein, thereby causing the plant cells to express the STY2 protein. Plants overexpressing the STY2 protein can be obtained by regenerating the plant cells. Preferably, the coding gene for the STY2 protein is transferred into the plant using Agrobacterium-mediated transformation.

[0083] In another embodiment of the present invention, an enhancing promoter or a tissue-specific promoter can be expressed to promote STY2 expression, or an enhancing promoter can be used to promote STY2 expression. Preferably, the STY2CDS region is introduced into plants using gene modification and gene transformation technologies. Promoters suitable for the method of the present invention include, but are not limited to, the 35S promoter, the Ubi promoter of rice and maize, etc.

[0084] Molecular markers, identification or screening

[0085] After learning about the function of STY2, it can be used as a molecular marker for targeted screening of plants.

[0086] Therefore, the present invention provides a use of the root nodule plant STY2 as a molecular marker for identifying root nodule traits, or as a molecular marker for targeted screening of plants; wherein the trait includes: nitrogen fixation capacity of the root nodule plant; preferably, the nitrogen fixation capacity of the root nodule plant includes: nodule infection cell formation, nodule development, red nodule development and maturation, nitrogen fixation capacity of the roots of the root nodule plant, nitrogen utilization rate of the root nodule plant, nitrogen fertilizer requirement of the root nodule plant, biomass of the root nodule plant, or yield of the root nodule plant.

[0087] After learning about the function of STY2, this new discovery can also be used to screen for substances or potential substances that can regulate the number of root nodules in rhizophytes by modulating this mechanism.

[0088] Therefore, the present invention also provides a method for selecting or identifying root nodule plants, the method comprising: identifying the expression of STY2 protein or its gene in a test plant; if the STY2 protein or its gene in the test plant is highly expressed or highly active, then it is a plant with high nitrogen fixation capacity; if the STY2 protein or its gene in the test plant is low expressed or low active, then it is a plant with low nitrogen fixation capacity; preferably, the nitrogen fixation capacity of the root nodule plant includes: nodule infection cell formation, nodule development, red nodule development and maturation, nitrogen fixation capacity of the roots of the root nodule plant, nitrogen utilization rate of the root nodule plant, nitrogen fertilizer requirement of the root nodule plant, biomass of the root nodule plant, or yield of the root nodule plant. This method can be applied to early identification, such as for the identification of plant seed / root tissue.

[0089] The present invention also provides a method for screening substances (potential substances) that regulate the nitrogen fixation capacity of rhizobium plants, comprising: (1) adding the candidate substance to a system expressing STY2; (2) detecting the system and observing the expression or activity of STY2 therein. If the expression or activity of STY2 is increased, it indicates that the candidate substance is a substance that can be used to increase the nitrogen fixation capacity of rhizobium plants; if the expression or activity of STY2 is decreased, it indicates that the candidate substance is a substance that can be used to reduce the nitrogen fixation capacity of rhizobium plants.

[0090] Methods for screening substances that act on proteins or genes or specific regions thereof as targets are well known to those skilled in the art, and these methods can all be used in this invention. The candidate substances can be selected from: peptides, polymeric peptides, peptide-like substances, non-peptide compounds, carbohydrates, lipids, antibodies or antibody fragments, ligands, small organic molecules, small inorganic molecules, and nucleic acid sequences, etc. Depending on the type of substance to be screened, those skilled in the art understand how to select an appropriate screening method.

[0091] A variety of conventional techniques can be used to identify gene transcription or expression in a system. These techniques include, but are not limited to, oligonucleotide hybridization (e.g., probes), polymerase chain reaction (PCR), and polyacrylamide gel electrophoresis. Detecting protein-protein interactions and their strength can be achieved using various techniques well-known to those skilled in the art, such as immunoprecipitation, GST precipitation, phage display, or yeast two-hybrid systems. Nuclear localization of proteins is also a well-known technique in the field.

[0092] In addition, the tobacco bimolecular fluorescence complementarity (BIFC) assay can also be used to analyze protein interactions. The principle is that fluorescent proteins (YFP, GFP, Luciferase, etc.) have many specific sites on their loop structures between the two β-sheets that allow for the insertion of exogenous proteins without affecting the fluorescent activity of the fluorescent protein. BiFC technology utilizes this characteristic of the fluorescent protein family, splitting the fluorescent protein into two non-fluorescent molecular fragments, which are then fused separately with target proteins for expression. If the two target proteins approach each other due to physical interactions, the two molecular fragments of the fluorescent protein spatially approach each other, reforming an active fluorescent group and emitting fluorescence.

[0093] Through large-scale screening, a class of potential substances that specifically act on STY2 or the signaling pathways involved by it can be obtained, which can regulate the number of root nodules in rhizobium plants.

[0094] The advantages of this invention include:

[0095] This invention reveals for the first time that STY2 is a transcription factor highly expressed in alfalfa root nodules, crucial for nodule development, especially the development of nitrogen-fixing red nodules. Further research shows that STY2 regulates the formation of nodule-infecting cells, and that promoter-driven STY2 gene expression can regenerate the mature red nodule phenotype that the sty2 mutant cannot form. Therefore, this gene can be used for plant improvement, enhancing the symbiotic nitrogen fixation efficiency of leguminous crops, and providing a theoretical basis and guidance for exploring symbiotic nitrogen fixation in non-leguminous crops.

[0096] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer.

[0097] Materials and methods

[0098] 1. Experimental Materials

[0099] 1.1 Plant materials

[0100] In this invention, Medicago truncatula R108 and A17 are used as wild types. The sty2 mutant is derived from the Noble Foundation's Tnt1 retrotransposon-tagged insertion mutant library. The spd1 mutant: EMS mutagenesis of wild-type alfalfa A17 resulted in an amino acid mutation at position 760 of the MtDMI1 protein, leading to the formation of spontaneous root nodules (Proc Natl Acad Sci USA 2022 Aug23; 119(34):e2205920119).

[0101] All materials were grown under the following conditions: 22°C, 16 hours of light, and 8 hours of darkness.

[0102] 1.2 Strains and Plasmid Vectors

[0103] 1.2.1 Strains

[0104] The strains used in this invention include the following:

[0105] The strain used for vector cloning was Escherichia coli DH10B, which was prepared using the highly efficient Escherichia coli heat-competent method.

[0106] The strain used for hair root transformation was Agrobacterium tumefaciens: AR1193; the strain used for tobacco injection was Agrobacterium GV3101 (pSoup-p19), purchased from Shanghai Weidi Biotechnology Co., Ltd.

[0107] The rhizobium strain (Sinorhizobium meliloti) used to inoculate alfalfa was Sm1021.

[0108] 1.2.2 Plasmid Vectors

[0109] The plasmids used in this invention include the following types:

[0110] Gateway system carrier: pDONR207 Gateway introductory carrier (Invitrogen).

[0111] Binary vectors used for plant transformation: pUB-GW-GFP, pK7WGF2, and pKGWFS7 are gene expression, protein localization, and tissue GUS expression vectors, respectively.

[0112] The Gateway system recombinase was purchased from Invitrogen, and the homologous recombinase was purchased from Nanjing Novizan Biotechnology Co., Ltd.

[0113] 2. Experimental Methods

[0114] 2.1. Construction of recombinant plasmids

[0115] First, using primers and high-fidelity DNA polymerase (purchased from Novizan), amplification was performed using alfalfa A17 genomic DNA as a template. The DNA was ligated into the pDONR207 entry vector via a BP reaction and transformed into E. coli. Positive clones were identified, plasmids were extracted, and after sequencing verification, they were ligated into the final vector via an LR reaction to obtain pK7WGF2-GFP-MtSTY2 and pKGWFS7-pMtSTY2-GUS plasmids, respectively.

[0116] The MtSTY2 gene of approximately 5 kb was amplified using primers. The PCR product was recovered and the pUB-GW-GFP vector was digested with PmeI and AscI enzymes. The vector was ligated with recombinase and transformed into E. coli to identify positive clones. The plasmid was extracted and sequenced for verification to obtain the pMtSTY2:STY2 vector.

[0117] The primer sequences are as follows:

[0118] MtSTY2-attB1-F: acaagtttgtacaaaaaagcaggcttcatggctggtttgttctcatt (SEQ ID NO: 3).

[0119] MtSTY2-attB2-R: accactttgtacaagaaagctgggttcaagatcttgggcgaggga (SEQ ID NO: 4).

[0120] pMtSTY2-attB1-F: acaagtttgtacaaaaaagcaggct actatcactagacgattttc (SEQ ID NO: 5).

[0121] pMtSTY2-attB2-R: accactttgtacaagaaagctgggt ccttaaccctaacaacaata (SEQ ID NO: 6).

[0122] MtSTY2-F: tgtcaaacactgatagtttaaacttccacatcttcttgtaga (SEQ ID NO: 7).

[0123] MtSTY2-R: tgaacgatcgatggcgcgcccggattgaagaatcacaata (SEQ ID NO: 8).

[0124] 2.2. Agrobacterium-mediated transformation

[0125] Step 1. Remove Agrobacterium competent cells from the -80℃ freezer, thaw them, add plasmids, and mix well;

[0126] Step 2. Transfer the competent cells to an electrode cup, place them in an electrostimulation apparatus for electrostimulation, and immediately add 500 μL of antibiotic-free LB liquid medium. Transfer the competent cells to a 1.5 mL centrifuge tube.

[0127] Step 3. Place the centrifuge tubes on a 28°C shaker to revive competent cells for 1 hour;

[0128] Step 4. Centrifuge at 4000 rpm for 5 min, resuspend approximately 50 μL of culture medium, and spread it evenly onto LB medium containing the corresponding antibiotic.

[0129] Step 5. Invert the culture medium and incubate it in a 28°C incubator for 2 days;

[0130] Step 6. Colony PCR identification, and pick positive clones.

[0131] 2.3. Plant root development transformation

[0132] Step 1. Use a pipette tip to pick up a small amount of straw and transfer it to LB liquid medium containing the corresponding antibiotic. Incubate at 28°C for 16-20 hours. On the second day, take 200 μL of bacterial culture and spread it evenly on LB solid medium containing the corresponding antibiotic. Incubate upside down for 1 day.

[0133] Step 2. Disinfect and germinate the alfalfa seeds. Place the swollen seeds in a refrigerator at 4°C overnight, and then germinate the seeds upside down overnight in the dark at 22°C.

[0134] Step 3. Use a yellow tube scraper to scrape the bacteria together on the plate, then use a sterilized scalpel to shorten the junction of the hypocotyl and radicle of the seed, leaving the upper part of the seed. Carefully apply Agrobacterium to the wound part of the seed with tweezers, then place it on MFP solid medium, seal it with sterile tape, and place it in a 22°C incubator;

[0135] Step 4. Remove the non-transformed roots of the plants that have grown in the incubator for 7 days, then transfer the plants to HRE solid medium with 200 mg / L termethin added, and incubate them in an incubator at 22°C for 10-14 days.

[0136] Step 5. Under a fluorescent stereomicroscope, cut off the non-transformed roots, leaving the fluorescent transformed roots;

[0137] Step 6: Transplant the selected seedlings into a 1:1 mixture of vermiculite and perlite, and inoculate with rhizobium (OD) 5 days later. 600 =0.02);

[0138] Step 7: After inoculating with rhizobia, water the plant every 2 days;

[0139] Step 8: 1-3 weeks after inoculation with rhizobia, remove the plant from the vermiculite, clean it, cut off the non-transformed roots, and begin to count the root nodule phenotype.

[0140] 2.2.4 Tissue semi-thin section

[0141] Step 1. Place fresh root nodules in FAA fixative and fix them. Use a vacuum dryer with program 1 to evacuate for about 1 hour. Discard the fixative, add fresh fixative, and place at 4°C overnight.

[0142] Step 2. Hardener-I solution preparation: Dissolve 1g of hardener-I powder in 100mL of Technovit 7100 base liquid (Technovit) and incubate overnight at 4°C;

[0143] Step 3. Discard the fixative and add 30%, 50%, 70%, 90%, and 100% ethanol sequentially for at least 30 minutes each to dehydrate the solution.

[0144] Step 4. Discard the ethanol and add 100% ethanol:Technovit 7100base liquid = 1:1 and let it permeate overnight;

[0145] Step 5. Discard the liquid and add pure Technovit 7100 base liquid for at least 30 minutes;

[0146] Step 6. Prepare a hardener-I:hardener-II mixture at a ratio of 15:1 and place it on ice. Add 200 μL of hardener to a 250 μL PCR tube. Carefully transfer the root nodules into the PCR tube using a toothpick, removing any air bubbles. After adjusting the position of the root nodules, tighten the cap to prevent air from entering and incubate overnight at 37°C.

[0147] Step 7. Use a semi-thin microtome to slice the resin-embedded material. Place the sliced ​​material on a glass slide with ice water dripped on it, carefully flatten it with tweezers, and then dry it with a 65°C slide dryer.

[0148] Step 8. Stain with 0.5% toluidine blue and observe and photograph under an optical microscope.

[0149] sequence

[0150] MtSTY2 gene sequence (SEQ ID NO:1)

[0151] ATGGCTGGTTTGTTCTCATTAGGAGGAGGTGGAAGAGGAAACCAAGGAG

[0152] AAGAATCACAACAACAAGGTCATATTCCACCACAAGAGACACTATTTTG

[0153] GTACAACAAAACGATGATGTTTCATCTTACAGAGGTAATTTAGAATTAT

[0154] GGAACCAACACCAACAACACCAGCAACATCAACAACAAGATGATATGC

[0155] ATGCTGCACGGCCGTTCTTCCCGGAGATCTCTACGGTCCAGGAGTTGGT

[0156] TTAGGTGTTGGACCAAGCAGGGTTCATCAGATGATCATGAACATCAAT

[0157] CGTCGTCGAGATCGGCGGCGTTACTATGGCGATGAGGTCTTCTTCTTCA

[0158] GCTGAAGGGATAAGTTGTCAAGATTGTGGTAACCAAGCTAAGAAAGATT

[0159] GTCCTCACATGAGATGTAGAACTTGTTGTAAGAGTCGTGGTTTTCAGTGT

[0160] CAAACTCATGTTAAAAGCACTTGGGTTCCTGCTTCTAGACGCCGTGAACG

[0161] TCAACAACAGTTATCTTCTTCTCCATTACAAAGAGATATTCTTAAACGCC

[0162] CTAGAGATGGTTCAAATGCTCTTGTTTCAACTCGTAATTTTCATACAGGT

[0163] ACTATTATATGAATGTGATGCATTTTTTTGTTGCTTTTTGTTTTTTAGTTAA

[0164] GGACTGGACTTGTACTGTGACATAAGGTGTAAGGTGTATTAATCAGAAAG

[0165] CACTAGCAGTTTCCGTGATTGACCCTTTTCAGTGTTATGGTTAAGAGAGT

[0166] GAGTGGTGAAATAAAGGAAAGCAAATACAAAGAGAGAATCTTTTGGTTT

[0167] AGAGGAATTCTCTGAAAAAGATAGCCAAATACTGATGACATAGTTTTGG

[0168] AACTATATCTGCTAACAATCCAACCGTATCGATTGCGTGTGTTTATGTTC

[0169] AAGCTTAAAATCTCCACCATATTTCATATCTATCAGCTTTTTTTTTCTTTT

[0170] TTTTTTTTTTATTTGTTTTGCATTTTCTTTGAAATGAAACCCTTTTTCTTTT

[0171] CTGAAAAACATCACTGTTATTTAGTAGCCCTGACCAAGAAAGAAACTGT

[0172] TTTTCCTCCTCTTTCTTAGCACATTCCCTCTCTCTTTCACATCCACACATA

[0173] TTCTCACCTCAATCACTAGCTACCATCATTTTCTTTGTGATTCTTTAATTC

[0174] TATGTTTAGATTATTCTAGAATATTAATTGCTTGCTATAGCTATAGCATA

[0175] AATTTTTGCATTCTGTTGTATTGTCGAGGTTAATTAATTAACATAGTCAA

[0176] ATAATATTATACAATATATGTCTAATGACTTTTTTGTTTTGTTAAAAAAT

[0177] AAAGGCTTAGAGGAAGCAAATTTTCCTGCTGTGGTAAGCTCACCAGCGG

[0178] AGTTCAGGTGTGTAAGGGTTAGTTCAATTGATGATGCAGATGATAGGTA

[0179] TGCATATCAAACGGCTGTTAACATTGGAGGACATTTGTTCAAAGGAATTC

[0180] TCTATGATTTTGGTCCAGAAAGTACCAACAATAGCAACAACAATAG

[0181] TAATTAATAATAGTAATTACATGATTGGGGAGACTTCCGGTGGAGGC

[0182] GGTGTTGGCGTTGCTCAACCGTTGAACCTAATTGCCGACTCTGATACCAC

[0183] TGTTGTTGCCTCTTCGGGCGCACTTGTTGATCCTTCTTCGTTGTATTCGGC

[0184] TCCGATTAATGCCTTCATGACGGCTAGTGGTACGCAATTCTTCCCTCGCC

[0185] CAAGATCTTGA

[0186] MtSTY2(SEQ ID NO:2)

[0187] MAGLFSLGGGGRGNQGEESQQQGHIPPQETLFWYNKNDDVSSYRGNLELW

[0188] NQHQQHQQHQQDDMHARPFFPRDLYGPGVGLGVGPSRVSSDDHEHQSS

[0189] SRSAAFTMAMRSSSAEGISCQDCGNQAKKDCPHMRCRTCCKSRGFQCQT

[0190] HVKSTWVPASRRRERQQLSSSPLQRDISKRPRDGSNALVSTRNFHTGLEEA

[0191] NFPAVVSSPAEFRCVRVSSIDDDRYAYQTAVNIGHLFKGILYDFGPESS

[0192] TNNSNNNSNYNNSNYMIGETSGGGGVGVAQPLNLIADSDTTVVASSGALVDPSSLYSAPINAFMTASGTQFFPRPRS*

[0193] Example 1: MtSTY2 is a transcription factor highly expressed in alfalfa root nodules.

[0194] The inventors previously obtained a spontaneous nodulation mutant, spd1, from *Alfalfa truncatula* using EMS mutagenesis. Transcriptome sequencing of spd1 nodules, combined with published RNA-Seq databases, identified MtSTY2 as a potential regulator of nodule development. Similar to genes related to nodule development, MtSTY2 expression is induced by rhizobia (…). Figure 1 A), subcellular localization of the protein shows that it is located in the cell nucleus ( Figure 1 B), using qRT-PCR verification, showed that MtSTY2 expression was low in roots but high in root nodules. Figure 1 C), tissue expression results of the MtSTY2 gene showed that it was highly expressed in the root nodule primordium and root nodule apex. Figure 1 D).

[0195] Example 2: MtSTY2 regulates root nodule organ development

[0196] To determine the role of MtSTY2 in root nodule organ development, the inventors ordered a Tnt1 insertion mutant of MtSTY2, Mtsty2, from the Noble Research Institute (USA) alfalfa seed mutant library. To clarify the genetic phenotype of Mtsty2, the inventors backcrossed it with the wild type. The mutant was then identified using gene-specific primers: P1: ATGGCTGGTTTGTTCTCATT (SEQ ID NO:9), P2: CTGTATGAAAATTACGAGTT (SEQ ID NO:10), and P3: ACAGTGCTACCTCCTCTGGATG (SEQ ID NO:11). qRT-PCR results showed that MtSTY2 expression was significantly reduced in the mutant, indicating that this is a gene knockout mutant. Figure 2 ).

[0197] To clarify the genetic phenotype of Mtsty2, the inventors inoculated the mutant with rhizobium S. meliloti1021 and observed and statistically analyzed the symbiotic nodulation at 1, 2, and 3 weeks after inoculation. The results showed that the Mtsty2 mutant plants exhibited a severe nitrogen deficiency phenotype: stunted aboveground parts and chlorotic leaves. Figure 3A). Further analysis of nodulation after 1-3 weeks of post-inoculation of the mutants showed that the number of root nodules formed by the Mtsty2 mutant was consistent with that of the wild type, while the number of nitrogen-fixing red nodules was reduced by 90%. Figure 3 C). These results indicate that MtSTY2 is crucial for root nodule development, but not for the initiation of root nodule organs.

[0198] Example 3: MtSTY2 regulates the formation of root nodule-infected cells

[0199] Phenotypic observation revealed that Mtsty2 could only form small white root nodules, but not pink root nodules with nitrogen-fixing capabilities. Figure 3 The results of nitrogenase activity also showed that the nitrogen fixation capacity of Mtsty2 root nodules was significantly reduced. Figure 4 C). To investigate why Mtsty2 root nodules cannot fix nitrogen, the inventors conducted semi-thin section observations of root nodules containing rhizobia at 10 dpi. The results showed that Mtsty2 root nodules could only form a small number of infected cells colonized by rhizobia. Figure 4 (AB) indicates that Mtsty2 root nodule cells cannot differentiate normally, leading to the failure of rhizobia to successfully colonize and the loss of nitrogen-fixing ability in the root nodules. Furthermore, the inventors found that the number of infection lines in Mtsty2 was not significantly different compared to the wild type. Figure 4 D). The above results indicate that MtSTY2 specifically regulates root nodule cell differentiation but does not participate in the rhizobium infection process.

[0200] Example 4: Genetic Complementation of the Mtsty2 Mutant

[0201] To further illustrate how MtSTY2 regulates root nodule development, the inventors conducted a genetic complementation experiment, transferring pMtSTY2:STY2 (an expression vector that uses the approximately 2kb promoter of STY2 to drive the STY2 gene) into Mtsty2.

[0202] The results showed that the expression of the MtSTY2 gene driven by its own promoter could compensate for the phenotype of the Mtsty2 mutant failing to form mature red root nodules. Figure 5 The number of red root nodules increased significantly.

[0203] The above results confirm that MtSTY2 plays an important role in the development of root nodule organs.

[0204] In summary, root nodules, as nitrogen-fixing sites, provide the microenvironment and energy required for nitrogen fixation by rhizobia, and normal root nodule development is a prerequisite for symbiotic nitrogen fixation. This invention, through analysis of the transcriptome of *Alfalfa truncatum* root nodules, identified a novel gene, MtSTY2, that regulates root nodule cell differentiation. MtSTY2 regulates root nodule differentiation to form nitrogen-fixing cells capable of being infected and colonized by rhizobia, providing a site and microenvironment for nitrogen fixation, thereby enabling symbiotic nitrogen fixation. Further research revealed that MtSTY2 specifically regulates the formation of root nodule nitrogen-fixing cells, and its own promoter-driven expression can compensate for the phenotype of the MtSTY2 mutant failing to form mature red root nodules. These findings have significant theoretical implications for understanding root nodule organ development and provide a theoretical basis and guidance for improving the efficiency of symbiotic nitrogen fixation in leguminous crops and exploring symbiotic nitrogen fixation in non-leguminous crops.

[0205] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims. Furthermore, all documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference.

Claims

1. A method for improving the nitrogen fixation capacity of rhizotrophs or a method for preparing rhizotrophs with increased nitrogen fixation capacity, comprising: Upregulate the expression or activity of STY2 in rhizobium plants.

2. The method as described in claim 1, characterized in that, Upregulation of STY2 expression or activity in plants includes: Introduce the STY2 coding gene or an expression construct or vector containing the coding gene into plants; Promote STY2 expression by expressing an enhancing promoter or a tissue-specific promoter; or, Enhancers promote STY2 expression; Preferably, the expression or activity of STY2 in rhizobia was upregulated in rhizobia with low or absent STY2 expression.

3. The use of a STY2 upregulator for improving the nitrogen fixation capacity of rhizobium plants.

4. The use as described in claim 3, characterized in that, The upregulators include: exogenous STY2 encoding genes or expression constructs or vectors containing such encoding genes; Preferably, the expression construct includes an enhanced promoter, a tissue-specific promoter, or an enhancer.

5. As described in any one of claims 1-4, characterized in that, The improvement of nitrogen fixation capacity of rhizobium plants includes: regulating the formation of rhizobium-infecting cells, promoting rhizobium development, promoting the development and maturation of red rhizobiums, improving the nitrogen fixation capacity of rhizobium roots, promoting the utilization of nitrogen by rhizobium plants, reducing the demand of rhizobium plants for nitrogen fertilizer, increasing the biomass of rhizobium plants, or increasing the yield of rhizobium plants.

6. As described in any one of claims 1-5, characterized in that, The root-nodule plants include or are selected from the following groups of plants: plants expressing the STY2 gene or protein, root-nodule plants, non-leguminous plants, grasses, and cruciferous plants; Preferably, the root nodule plant is a leguminous plant; More preferably, the legumes include: Tribulus terrestris, alfalfa, tribulus terrestris alfalfa, soybean, broad bean, pea, mung bean, red bean, cowpea, common bean, hyacinth bean, pigeon pea, peanut, milkvetch, broad bean, sagebrush, albizia, rosewood, soapberry, ebony, red bean, pagoda tree, horsethorn, pagoda tree flower, indigo, sappanwood, gum, gum arabic, astragalus gum, copal gum, Indian hemp, kudzu, and spider vine.

7. As described in any one of claims 1-6, characterized in that, The amino acid sequence of the STY2 protein is selected from the following group: (i) A protein having the amino acid sequence shown in SEQ ID NO:2; (ii) A protein derived from (i) having the regulatory trait function formed by substituting, deleting or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID NO:2; (iii) A protein whose amino acid sequence has ≥80% homology with the amino acid sequence shown in SEQ ID NO:2 and has the function of regulating the aforementioned traits; (iv) The active fragment of the polypeptide with the amino acid sequence shown in SEQ ID NO:2; or, (v) A protein formed by adding a tag sequence or restriction enzyme site sequence to the N or C end of a polypeptide with the amino acid sequence shown in SEQ ID NO:2, or by adding a signal peptide sequence to its N end.

8. The use of a root nodule plant, STY2, as a molecular marker for identifying root nodule plant traits, or as a molecular marker for targeted plant screening; wherein, The traits include: nitrogen fixation capacity of rhizobium plants; Preferably, the nitrogen fixation capacity of the root nodule plant includes: nodule infection cell formation, nodule development, red nodule development and maturation, nitrogen fixation capacity of the roots of the root nodule plant, nitrogen utilization rate of the root nodule plant, nitrogen fertilizer requirement of the root nodule plant, biomass of the root nodule plant, or yield of the root nodule plant.

9. A method for selecting or identifying root-nodule plants, characterized in that, The method includes: identifying the expression of STY2 protein or its gene in the test plant; If the STY2 protein or its gene is highly expressed or highly active in the test plant, it is a plant with high nitrogen fixation capacity; if the STY2 protein or its gene is low expressed or low active in the test plant, it is a plant with low nitrogen fixation capacity. Preferably, the nitrogen fixation capacity of the root nodule plant includes: nodule infection cell formation, nodule development, red nodule development and maturation, nitrogen fixation capacity of the roots of the root nodule plant, nitrogen utilization rate of the root nodule plant, nitrogen fertilizer requirement of the root nodule plant, biomass of the root nodule plant, or yield of the root nodule plant.

10. A method for screening substances that regulate the nitrogen fixation capacity of rhizobium plants, comprising: (1) Add the candidate substance to the system expressing STY2; (2) Detect the system and observe the expression or activity of STY2. If the expression or activity of STY2 is increased, it indicates that the candidate substance is a substance that can be used to increase the nitrogen fixation capacity of rhizobium plants; if the expression or activity of STY2 is decreased, it indicates that the candidate substance is a substance that can be used to reduce the nitrogen fixation capacity of rhizobium plants.

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