Identification of HHO1 and application of HHO1 in symbiotic nitrogen fixation of leguminous plants

Abstract

The invention provides identification of HHO1 and application of HHO1 in symbiotic nitrogen fixation of leguminous plants. The gene participating in the symbiotic process of root nodules is separated from leguminous plants for the first time, is a transcription factor, can regulate the nitrogen fixation character of the leguminous plants, and is named as HHO1 by the inventor. By optimizing the expression level of HHO1, the symbiosis of root nodules can be regulated and controlled, and the characters of leguminous plants can be optimized. The invention provides a new way for variety improvement of leguminous plants.

Description

Technical Field

[0001] This invention belongs to the field of plant molecular biology and plant genetic engineering. More specifically, this invention relates to the identification of HHO1 and its application in symbiotic nitrogen fixation in legumes. Background Technology

[0002] Nitrogen is one of the three essential mineral elements for plant growth and development, participating in numerous physiological and biochemical processes. Nitrogen primarily originates from atmospheric nitrogen gas and is a crucial nutrient element for plant growth and development. However, this abundant nitrogen gas cannot be directly absorbed and utilized by plants; they can only absorb nitrate nitrogen or ammonia nitrogen from the environment. Nitrogen fixation mainly occurs through two mechanisms: chemical nitrogen fixation and biological nitrogen fixation. In the past, people produced and applied large quantities of industrial nitrogen fertilizers to increase crop yields. While industrial nitrogen fixation can produce the nitrogen fertilizer needed by plants, the excessive use of nitrogen fertilizers severely damages the natural environment.

[0003] Therefore, beyond industrial nitrogen fertilizers, researchers are increasingly focusing on environmentally friendly biological nitrogen fixation. This highly efficient and environmentally friendly method plays a significant role in practical production. Biological nitrogen fixation primarily involves nitrogen-fixing microorganisms reducing atmospheric nitrogen into ammonia; thus, nitrogen-fixing organisms can be considered the largest natural nitrogen fertilizer processing plant on Earth. Among these, the symbiotic nitrogen fixation between legumes and rhizobia plays a crucial role in biological nitrogen fixation. Studying the symbiotic interaction between legumes and nitrogen-fixing rhizobia is of great significance for the production of both food and cash crops.

[0004] Symbiotic nitrogen fixation between legumes and rhizobia is a mutually beneficial process, with both sides possessing sophisticated regulatory mechanisms. The host plant provides rhizobia with carbohydrates—products of photosynthesis—which are essential for the rhizobia's growth; conversely, the rhizobia reduce atmospheric nitrogen to ammonia in the root nodules, providing the host plant with the nitrogen source necessary for its growth and development. The basic process of root nodule formation in legumes includes: 1) Early molecular dialogue between legumes and rhizobia: Legume roots secrete flavonoids into the soil, which are sensed by specific rhizobia and activate the expression of the rhizobia's transcription factor NodD. NodD then activates the expression of NodA, B, and C, synthesizing a polysaccharide signaling molecule called the nodfactor. 2) The nodfactor receptors (LjNFR1, LjNFR5) in the plant sense and recognize the nodfactor signal, thereby activating both rhizobia infection and root nodule organ formation. 3) Rhizobia attach to the tips of root hairs, causing the root hairs to swell and deform, and envelop the rhizobia. Subsequently, the host plant plasma membrane invaginates, forming a tubular structure - the infection line. The rhizobia extend along the infection line from the epidermal cells to the cortical cells, and finally reach the root nodule primordium, releasing the rhizobia from the infection line to form a nitrogen-fixing bacterium-like body.

[0005] In this field, several regulatory mechanisms and related regulatory genes have been identified for symbiotic nitrogen fixation. However, recent years have also revealed that the number of effective root nodules is a crucial factor in achieving optimal nitrogen utilization and corresponding development, growth, and fruiting in rhizomatous plants. A higher number of effective root nodules is not necessarily better; the nodulation process consumes energy, and excessive nodulation can lead to stunted growth due to excessive energy expenditure. Given that mycorrhizal nodulation is a balanced process, both excessive and insufficient nodules can be detrimental. Therefore, identifying regulatory molecules that increase or decrease the number of nodules is of significance. Summary of the Invention

[0006] The purpose of this invention is to provide an identification method for HHO1 and its application in symbiotic nitrogen fixation in legumes. This invention marks the first time that a novel gene, HHO1, with a specific function has been cloned from legumes, and the molecular mechanism by which it controls mycorrhizal symbiosis has been analyzed, providing technical support for creating breeding materials suitable for efficient nitrogen fixation in legumes.

[0007] In a first aspect of the invention, an use of HHO1 is provided for regulating nitrogen fixation traits in legumes, or for use as a regulatory target to prepare regulatory molecules that regulate nitrogen fixation traits in legumes; the nitrogen fixation traits include: root nodule number, rhizobium infection, and nitrogenase activity.

[0008] In one or more embodiments, the regulatory molecule is an HHO1 upregulated (overexpressed) molecule that reduces rhizobium infection (reduces infection events), reduces the number of root nodules in plants, and reduces nitrogenase activity; preferably, the upregulated molecule includes: an exogenous HHO1 encoding gene or an expression construct or vector containing the encoding gene.

[0009] In one or more embodiments, the terms "upgrade", "promote", "enhance" or "increase" indicate a significant upgrade, promotion, enhancement or increase, such as an upgrade, promotion, enhancement or increase of 5%, 10%, 20%, 40%, 60%, 80%, 100%, 200%, 300%, 500%, 800% or higher.

[0010] In one or more embodiments, the reduction of rhizobium infection includes: significantly reducing infection points (Foci), significantly reducing infection lines (IT), and significantly reducing root nodule primordia (NP).

[0011] In one or more embodiments, nitrate treatment is used to induce the expression of HHO1.

[0012] In one or more embodiments, the regulatory molecule is an HHO1 downregulatory molecule that increases the number of root nodules in plants; preferably, the downregulatory molecule includes: a reagent for performing a loss-of-function mutation on HHO1, a reagent for knocking out or silencing HHO1, a reagent for inhibiting HHO1 activity; preferably, it includes: an interfering molecule that specifically interferes with the expression of the gene encoding HHO1; a CRISPR gene editing reagent, a homologous recombination reagent, or a site-directed mutagenesis reagent targeting HHO1.

[0013] In one or more embodiments, the downregulated molecule is an interfering molecule comprising RNAi formed by primers of the nucleotide sequences shown in SEQ ID NO:8 and SEQ ID NO:9 (e.g., annealed to form a double strand).

[0014] In one or more embodiments, the downregulation, suppression, or reduction refers to a significant downregulation, suppression, or reduction, such as a downregulation, suppression, or reduction of 5%, 10%, 20%, 40%, 60%, 80%, 100%, 200%, 300%, 500%, 800%, or higher.

[0015] In one or more embodiments, the downregulated expression includes missing expression.

[0016] In one or more embodiments, the amino acid sequence of the HHO1 polypeptide is selected from the group consisting of:

[0017] (i) A polypeptide having the amino acid sequence shown in SEQ ID NO:1;

[0018] (ii) A polypeptide having the regulatory trait function, with an amino acid sequence homology ≥80% (preferably ≥85%, ≥90%, ≥95% or ≥98%) to the amino acid sequence shown in SEQ ID NO:1;

[0019] (iii) A polypeptide derived from (i) having the regulatory trait function, formed by substituting, deleting, or adding one or more (e.g., 1-20, 1-10, 1-5, 1-3) amino acid residues of the amino acid sequence shown in SEQ ID NO:1; or,

[0020] (iv) A polypeptide formed by adding a tag sequence or restriction enzyme site sequence to the N or C end of the polypeptide with the amino acid sequence shown in SEQ ID NO:1, or by adding a signal peptide sequence to its N end.

[0021] In another aspect of the present invention, a method for regulating nitrogen-fixing traits in legumes is provided, comprising: regulating (selected from increasing or decreasing) the expression or activity of HHO1 in legumes; wherein the nitrogen-fixing traits include: root nodule number, rhizobium infection, and nitrogenase activity.

[0022] In one or more embodiments, increasing the expression or activity of NHHO1 in plants, or increasing the expression or activity of HHO1 in plants with low HHO1 expression, thereby reducing rhizobium infection (reducing infection events), reducing the number of root nodules in plants, and reducing nitrogenase activity; the method includes (but is not limited to): introducing the HHO1 coding gene or an expression construct or vector containing the coding gene into plants; performing gain-of-function mutations on HHO1 (e.g., for plants with low HHO1 function / activity); promoting HHO1 expression by expressing an enhancing promoter or a tissue-specific promoter; or promoting HHO1 expression by an enhancer.

[0023] In one or more embodiments, the expression or activity of HHO1 in plants is downregulated, thereby increasing the number of root nodules in the plants; the method includes (but is not limited to): performing a loss-of-function mutation on HHO1 in plants containing HHO1, knocking out or silencing the gene encoding HHO1, or inhibiting the activity of HHO1.

[0024] In one or more embodiments, the loss-of-function mutation of HHO1 includes: targeted modification of the HHO1 coding gene to reduce its function / activity; preferably, causing a frameshift in its coding gene to prematurely terminate translation. Preferably, causing a frameshift in its coding gene to prematurely terminate translation; for example, inserting a foreign gene fragment into the HHO1 gene, disrupting its coding sequence to downregulate HHO1, or inactivating it.

[0025] In one or more embodiments, the expression of the HHO1 coding gene is interfered with by interfering molecules, such as (but not limited to) siRNA, shRNA, miRNA, antisense nucleotides, etc. Preferably, the interfering molecule comprises RNAi formed by primers (e.g., annealing to form a double strand) of the nucleotide sequences shown in SEQ ID NO:8 and SEQ ID NO:9.

[0026] In one or more embodiments, for the purpose of downregulating HHO1 in plants, a plant screening library formed by gene insertion deletion, mutation deletion, or deletion deletion can be constructed, from which plants (mutants) with downregulated HHO1 can be screened.

[0027] In one or more embodiments, the method includes: performing gene editing using a CRISPR system to knock out the coding gene for HHO1.

[0028] In one or more embodiments, the method includes: silencing HHO1 with an interfering molecule that specifically interferes with the expression of the gene encoding HHO1.

[0029] In one or more embodiments, the coding gene for HHO1 is knocked out by homologous recombination.

[0030] In one or more embodiments, after preparing legumes with altered nitrogen-fixing traits using the method described above, the method further includes: hybridizing the plant with regulated HHO1 expression or activity with a plant without HHO1 peptide or its encoding gene to obtain hybrid offspring.

[0031] In one or more embodiments, the legumes include (but are not limited to): alfalfa, soybean, birdsfoot root, pea, peanut, kidney bean, mung bean, adzuki bean, broad bean, cowpea, or milkvetch.

[0032] In one or more embodiments, the HHO1 includes a cDNA sequence, a genomic sequence (gDNA), or a sequence that is artificially optimized or modified based on them.

[0033] In one or more embodiments, the HHO1 is derived from legumes.

[0034] In one or more embodiments, the HHO1 is derived from alfalfa.

[0035] In one or more embodiments, the HHO1 includes its homologs.

[0036] In another aspect of the present invention, a use is provided for HHO1, a legume, as a molecular marker for identifying nitrogen-fixing traits in legumes, or as a molecular marker for targeted screening of plants; wherein the nitrogen-fixing traits include: root nodule number, rhizobium infection, and nitrogenase activity.

[0037] In one or more embodiments, the expression or sequence characteristics of HHO1 protein or its gene in the test plant are identified; if the test plant has high expression (including normal expression) or high activity (including normal expression) of HHO1 protein or its gene, it is a plant with reduced rhizobium infection, fewer root nodules, and lower nitrogenase activity (compared to the average level of this type of plant); if the test plant has low expression (including no expression) or low activity (including no activity) of HHO1 protein or its gene, it is a plant with a high number of root nodules (compared to the average level of this type of plant).

[0038] In one or more embodiments, high expression or high activity refers to a statistically significant increase in expression or activity compared to the average expression or activity of similar or identical plants.

[0039] In one or more embodiments, the low expression or low activity refers to a statistically significant reduction in expression or activity compared to the average expression or activity of control plants (including plants of the same type or species).

[0040] In another aspect of the invention, a legume cell, tissue, or organ (including root nodule cells, root nodule organs, or root nodule tissue) is provided, wherein the cell contains an exogenous regulatory molecule of HHO1.

[0041] In one or more embodiments, the regulatory molecule is an HHO1 upregulatory molecule, comprising: an exogenous HHO1 encoding gene or an expression construct or vector containing the encoding gene; or, the regulatory molecule is an HHO1 downregulatory molecule, comprising: a reagent for loss-of-function mutation of HHO1, a reagent for knocking out or silencing HHO1, a reagent for inhibiting HHO1 activity; preferably comprising: an interfering molecule that specifically interferes with the expression of the HHO1 encoding gene; a CRISPR gene editing reagent, a homologous recombination reagent, or a site-directed mutagenesis reagent targeting HHO1.

[0042] In another aspect of the present invention, a method is provided for screening substances (including potential substances) that regulate nitrogen fixation traits (phenotypes) in leguminous plants, comprising: (1) adding a candidate substance to a test system containing HHO1; (2) detecting the system and observing the expression or activity of HHO1 therein; if the expression or activity of HHO1 is increased (statistically increased, such as by 10%, 20%, 40%, 60%, 80%, 90% or higher), it indicates that the candidate substance is a substance that can be used to reduce rhizobium infection (reduce infection events), reduce the number of root nodules in plants, and reduce nitrogenase activity; if the expression or activity of HHO1 is decreased (statistically decreased, such as by 10%, 20%, 40%, 60%, 80%, 90% or lower), it indicates that the candidate substance is a substance that can be used to increase the number of root nodules in plants.

[0043] In one or more embodiments, the expression construct includes an enhancing promoter, a tissue-specific promoter, or an enhancer; or, a reagent for gain-of-function point mutation of HHO1.

[0044] In one or more embodiments, the screening method further includes setting up a control group to clearly distinguish the difference between HHO1 expression or activity in the test group and the control group.

[0045] In one or more embodiments, the candidate substances include (but are not limited to): regulatory molecules (such as upregulatory molecules, small molecule compound gene editing constructs, etc.) designed for HHO1 or its encoding gene or their upstream or downstream proteins or genes.

[0046] Other aspects of the invention will be apparent to those skilled in the art from the disclosure herein. Attached Figure Description

[0047] Figure 1 HHO1 expression pattern.

[0048] (A) Samples were taken at different time points (12h, 1d, 3d, 5d, 7d, 10d, 14d) after inoculation with rhizobium to examine the expression of HHO1 in the R108 ecotype and nin-2 mutant.

[0049] (B) Samples were taken before and after 3 hours of treatment with 5mM KNO3 to examine the expression of HHO1 in the R108 ecotype and the nlp1-1 mutant.

[0050] (C) Expression of HHO1 promoter fusion GUS in root hairs and root nodules, in which the expression of HHO1 promoter fusion GUS in root nodules at different time points (5d, 7d, 10d, 14d) was observed.

[0051] (D) After treatment with 5mM KNO3 for 30 min, the expression of HHO1 in the root was enhanced.

[0052] Figure 2 Statistical analysis of symbiotic phenotypes of HHO1 overexpression.

[0053] (AD)HHO1 overexpression leads to infection and nodulation phenotypes in rhizobia.

[0054] Figure 3 Statistics on the symbiotic phenotypes of HHO1 RNAi.

[0055] (AC)HHO1 RNAi, nodulation phenotype of rhizobia inoculated with rhizobia.

[0056] Different letters indicate statistical differences between experimental groups, one way ANOVA (multiple comparisons). Detailed Implementation

[0057] This invention proposes a novel target for regulating nodulation traits in leguminous plants. Through in-depth research and screening, and correlation analysis of various experimental results, the inventors isolated a gene involved in the root nodule symbiosis process from *Alfalfa*, a leguminous plant. This gene, a transcription factor, regulates nitrogen fixation in leguminous plants and has been named HHO1. By optimizing the expression level of HHO1, root nodule symbiosis can be regulated, thereby guiding agricultural production, reducing fertilizer and labor inputs, optimizing traits, and improving economic benefits. This invention provides a new approach for the variety improvement of leguminous plants.

[0058] In this invention, the term "optimized traits" or "improved traits" refers to improving the characteristics of legumes, such as regulating nitrogen fixation traits in legumes.

[0059] In this invention, the term "leguminous plants" includes plants that express HHO1. Based on knowledge in the art, plants containing HHO1 or its homologs possess the mechanisms claimed in this invention, thus enabling the anticipation and achievement of the technical effects described herein.

[0060] In this invention, the "leguminous plant" is a type of "nodule plant," which is a plant that can be invaded and stimulated by rhizobia to form nodules on its roots. The "nodule plant" can include leguminous nodule plants and non-leguminous nodule plants. Preferably, the "nodule plant" is a "leguminous plant" (a leguminous plant capable of forming a symbiotic nitrogen-fixing relationship with rhizobia). Preferably, the "leguminous plant" includes "alfalfa".

[0061] "Nodule plants" also include "nodule-like plants," which refers to plants with nodule-like or root-nodule-like structures.

[0062] The term "control plant" is used in this invention. Selecting a suitable control plant is a routine part of the experimental design and may include a corresponding wild-type plant or a corresponding transgenic plant without the target gene. Control plants are generally the same plant species or even varieties of the same or the same class as the plant being evaluated. Control plants can also be individuals that have lost their transgenic components due to segregation. As used herein, control plants refer not only to whole plants but also to plant parts, including seeds and seed portions.

[0063] In this invention, "exogenous" or "heterogeneous" refers to the relationship between two or more nucleic acid or protein sequences from different sources. For example, if the combination of a promoter and a target gene sequence is not naturally occurring, then the promoter is exogenous to the target gene. A specific sequence is "exogenous" to the cell in which it is inserted.

[0064] In this invention, "introduction" or "transformation" includes transferring exogenous polynucleotides into host cells, and there are no particular limitations on the methods of "introduction" or "transformation" in this invention.

[0065] HHO1 gene

[0066] Existing research in this field has not reported the correlation between the HHO1 gene and root nodule regulation in legumes. The inventors have cloned the symbiotic interaction receptor kinase gene HHO1 from alfalfa for the first time, and studies have shown that it is closely related to nitrogen fixation traits in legumes. This invention elucidates for the first time the molecular mechanism by which the HHO1 gene participates in regulating nitrogen fixation traits in legumes.

[0067] In this invention, HHO1 refers to a polypeptide having the sequence of SEQ ID NO:1 or its encoding gene (HHO1 gene), and also includes sequence variations having the same function as the HHO1 polypeptide. The encoding gene can be gDNA or cDNA, and may also contain a promoter. For example, the cDNA has the nucleotide sequence shown in SEQ ID NO:2. The sequence of the encoding gene also includes sequences degenerate with the sequences provided in this invention.

[0068] Variations of the HHO1 polypeptide include (but are not limited to): deletions, insertions, and / or substitutions of several amino acids (e.g., 1-40, preferably 1-30, more preferably 1-20, most preferably 1-10, and even more preferably 1-8 or 1-5), and additions or deletions of one or more amino acids (e.g., up to 20, preferably up to 10, and more preferably up to 5) at the C-terminus and / or N-terminus. Any protein with high homology to the HHO1 polypeptide (e.g., 80% or higher homology to the polypeptide sequence shown in SEQ ID NO:1; preferably 85% or higher; more preferably 90% or higher, such as 95%, 98%, or 99% homology) and possessing the function of regulating nitrogen-fixing traits in legumes should also be included in this invention.

[0069] In addition, polypeptides derived from other leguminous plants besides alfalfa that have high homology with the sequence shown in SEQ ID NO:1, or that play the same or similar roles in the same or similar regulatory pathways, are also included in this invention.

[0070] The present invention also includes mutant forms of HHO1 or truncated polypeptide fragments, provided that they retain the activity of the full-length protein in regulating nitrogen fixation traits in legumes.

[0071] In this invention, HHO1 also includes its homologs. That is, other polypeptides or genes obtained from other species that are highly homologous to HHO1 (e.g., having more than 60%, such as 70%, 80%, 85%, 90%, 95%, or even 98% sequence identity) are also within the scope of this invention.

[0072] The polynucleotide (gene) encoding the HHO1 polypeptide can be a natural gene from legumes or a degenerate sequence thereof, as will be understood by those skilled in the art.

[0073] 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.

[0074] The host cell can be 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 embryonic transformation method; the Agrobacterium-mediated transformation is preferred. Transformed plant cells, tissues, or organs can be regenerated into plants using certain methods, thereby obtaining plants with altered traits compared to the wild type. However, this invention also covers some cells, tissues, or organs that cannot be directly regenerated into plant varieties.

[0075] application

[0076] The inventors discovered, through detecting the transcriptional level of HHO1, that HHO1 is induced by rhizobia, and this process depends on the symbiotic-specific transcription factor NIN. Furthermore, this process is associated with the transcription factor NLP1. Further observation of the symbiotic phenotype of HHO1 overexpression through root transformation revealed that, under rhizobia inoculation, HHO1 overexpression exhibited a significantly reduced phenotype in infection and nodulation, demonstrating that HHO1 plays a crucial role in regulating nodulation in leguminous plants.

[0077] In this invention, the HHO1 promoter of *Alfalfa tribulus* was isolated, as shown in SEQ ID NO:3. Through promoter fusion with GUS expression experiments, the expression pattern of HHO1 at different time points (5 days, 7 days, 10 days, and 14 days) after inoculation with rhizobia was determined; the induced expression pattern of HHO1 after nitrate treatment was also determined. This invention elucidates the function of HHO1 in root nodule symbiosis by constructing a root-transforming line overexpressing HHO1.

[0078] Based on the inventor's new discovery, this invention provides a method for regulating nitrogen-fixing traits (phenotypes) in legumes or for preparing legumes with changes in nitrogen-fixing traits (phenotypes), comprising: regulating the expression or activity of HHO1 in legumes. Depending on the plant's need for nitrogen fixation in root nodules, related regulatory actions are implemented to achieve the most ideal phenotype possible and efficiently utilize nitrogen. On the one hand, by upregulating the expression or activity of HHO1 in plants, or increasing the expression or activity of HHO1 in plants with low HHO1 expression, the number of root nodules is reduced, thereby reducing rhizobium infection (reducing infection events), decreasing the number of root nodules, and lowering nitrogenase activity. On the other hand, by downregulating the expression or activity of HHO1 in plants, the number of root nodules is increased.

[0079] This invention provides a method for upregulating HHO1 expression in plants. The method includes: transferring the HHO1 coding gene or an expression construct (including an expression cassette) or vector containing the coding gene into plants. Alternatively, gain-of-function mutations can be performed on HHO1 or its coding gene; expression of the HHO1 coding gene can be promoted by expressing an enhancing promoter or a tissue-specific promoter; or, expression of the HHO1 coding gene can be promoted by an enhancer. It should be understood that other methods for upregulating HHO1 expression in plants should also be included in this invention.

[0080] In this invention, the HHO1 upregulating molecules include agonists, promoters, and stimulants, and these terms are used interchangeably. The HHO1 upregulating molecules refer to any substance that can increase HHO1 activity, enhance HHO1 stability, upregulate HHO1 expression, increase the effective duration of HHO1 action, or promote the transcription and translation of the HHO1 gene. These substances can all be used in this invention as substances useful for upregulating HHO1, thereby exerting a regulatory effect. They can be biomolecules, compounds, small chemical molecules, etc. The biomolecules can be at the nucleic acid level (including DNA and RNA) or at the protein level.

[0081] In this invention, the downregulating molecule for the HHO1 protein or its encoding gene refers to any substance that can reduce the activity of the HHO1 protein, reduce the stability of the HHO1 protein or its encoding gene, downregulate the expression of the HHO1 protein, reduce the effective duration of the HHO1 protein, inhibit the transcription and translation of the HHO1 gene, or reduce the phosphorylation / activation level of the protein. These substances can all be used in this invention as substances useful for downregulating HHO1. They can be biomolecules, compounds, small chemical molecules, etc. The biomolecules can be at the nucleic acid level (including DNA and RNA) or at the protein level. For example, the downregulating molecule is: an interfering RNA molecule or antisense nucleotide that specifically interferes with the expression of the HHO1 gene; or a gene editing reagent that specifically edits HHO1, etc.

[0082] The present invention also provides a method for downregulating HHO1 in plants, comprising: performing a loss-of-function mutation on HHO1 in plants containing HHO1, knocking out or silencing the coding gene of HHO1, or inhibiting the activity of HHO1; preferably, the loss-of-function mutation on HHO1 comprises: targeting and modifying the coding gene of HHO1 to reduce its function / activity; preferably, causing a frameshift in its coding gene to terminate translation prematurely.

[0083] As a relatively specific implementation method, gene editing can be performed using the CRISPR / Cas9 system to knock out or downregulate target genes. Suitable sgRNA target sites lead to higher gene editing efficiency, so appropriate target sites can be designed and identified before starting gene editing. After designing specific target sites, in vitro cell activity screening is also necessary to obtain effective target sites for subsequent experiments.

[0084] As a relatively specific embodiment, the downregulator can be an HHO1 gene-specific interfering RNA molecule (such as siRNA, shRNA, miRNA, etc.). The interfering RNA can be delivered into cells using appropriate transfection reagents, or it can be delivered into cells using various techniques known in the art.

[0085] In some embodiments, RNAi is used to suppress the HHO1 gene. RNAi is an evolutionarily conserved cellular defense mechanism used to control the expression of exogenous genes in most eukaryotes, including humans. RNAi is typically triggered by double-stranded RNA (dsRNA) and induces sequence-specific mRNA degradation of the single-stranded target RNA. The mediators of mRNA degradation are small interfering RNA duplexes (siRNAs), which are typically produced by the enzymatic cleavage of long dsRNA within the cell. siRNAs are typically about 21 nucleotides long (e.g., 21–23 nucleotides). After the small RNA or RNAi is introduced into the cell, the sequence is believed to be delivered to an enzyme complex called the RISC (RNA-induced silencing complex). The RISC recognizes the target and cleaves it with a nuclease. Notably, if a larger RNA sequence is delivered to the cell, the RNase III enzyme (Dicer) converts the longer dsRNA into a 21–23 nt ds-siRNA fragment.

[0086] In some embodiments, shRNA technology is used for interference. shRNA is an RNA sequence that can rotate a tight hairpin, which can be used to silence gene expression via RNA interference. shRNA uses a vector to introduce it into the cell and utilizes a promoter (such as U6) to ensure that the shRNA is always expressed. This vector is typically delivered to daughter cells, allowing gene silencing to be inherited. The shRNA hairpin structure is cleaved into siRNA by cellular mechanisms and then binds to the RNA-induced silencing complex (RISC). This complex binds to and cleaves mRNAs that match the bound siRNA. shRNA is transcribed by RNA polymerase III.

[0087] In a preferred embodiment, an RNAi molecule formed from primers containing the nucleotide sequences shown in SEQ ID NO:8 and SEQ ID NO:9 is provided, which provides an appropriate knockdown effect, has no visible toxicity to the body, but is particularly effective in inhibiting metastatic colon cancer.

[0088] As an optional implementation, HHO1 expression is modulated using antisense compounds that specifically hybridize with one or more nucleic acids encoding HHO1. The specific hybridization of oligomers with their target nucleic acids interferes with the normal function of the nucleic acids. This modulation of target nucleic acid function by compounds that specifically hybridize with the target nucleic acid is commonly referred to as "antisense."

[0089] It should be understood that the methods for downregulating target genes / proteins in plants are not limited to those listed above. Once the functions of HHO1 and the signaling pathways it participates in (preferably including its upstream and downstream genes) are known, various methods well-known to those skilled in the art can be used to regulate the expression or activity of HHO1 or to regulate related upstream or downstream genes of HHO1. For example, various methods well-known to those skilled in the art can be used to overexpress HHO1 or its upstream or downstream genes.

[0090] The present invention also provides a method for screening substances (potential substances) that regulate nitrogen fixation traits (phenotypes) in legumes, comprising: (1) adding a candidate substance to a test system containing HHO1; (2) detecting the system and observing the expression or activity of HHO1 therein; if the expression or activity of HHO1 is increased (statistically increased), it indicates that the candidate substance is a substance that can be used to reduce rhizobium infection (reduce infection events), reduce the number of root nodules in plants, and reduce nitrogenase activity; if the expression or activity of HHO1 is decreased (statistically decreased), it indicates that the candidate substance is a substance that can be used to increase the number of root nodules in plants.

[0091] 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.

[0092] 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 techniques (such as 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. Protein localization is also a well-known technique in the field.

[0093] 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.

[0094] Through large-scale screening, a class of potential substances that specifically act on HHO1 or the signaling pathways it participates in can be obtained, which have a regulatory effect on nitrogen fixation traits in legumes.

[0095] Furthermore, after learning about the function of HHO1, it can be used as a molecular marker for screening superior plant varieties.

[0096] After learning about the function of HHO1, this new discovery can also be used to screen for substances or potential substances that can regulate nitrogen fixation traits (phenotypes) in legumes by modulating this mechanism.

[0097] According to a new discovery of the present invention, a method for targeted selection or identification of plants is provided, comprising: identifying the expression or sequence characteristics of HHO1 protein or its gene in a test plant; if the HHO1 protein or its gene in the test plant is highly expressed or highly active, it is characterized by reduced rhizobium infection, fewer root nodules, and lower nitrogenase activity (compared to the average level of this type of plant); if the HHO1 protein or its gene in the test plant is low expressed (including not expressed) or low active (including inactive), it is characterized by a high number of root nodules (compared to the average level of this type of plant). This method can be applied to early identification, such as for the identification of plant seed / root tissue.

[0098] The mutually beneficial symbiotic relationship between microorganisms and plants provides plants with ample nutrients, contributing to their growth and development, thereby increasing crop yield and quality. Root nodule symbiosis is a natural and environmentally friendly nitrogen fixation method, with lower energy consumption and less environmental pollution compared to industrially synthesized nitrogen fertilizers. Mycorrhizal symbiosis has many positive effects on plants, including improving nutrient absorption capacity, enhancing stress resistance, promoting growth and development, and promoting ecosystem balance. Therefore, this invention provides insights into mycorrhizal symbiosis that can help further explore molecules with regulatory functions and optimize them through genetic engineering to improve the symbiotic range and nutrient absorption efficiency of leguminous plants, thereby promoting the sustainable development of agricultural production.

[0099] The HHO1 gene of this invention plays a crucial role in root nodule symbiosis. It provides a new approach to optimizing nodule number and nodule-forming ability in legumes. Furthermore, since HHO1 is also a nitrogen-inducible transcription factor, its nodule-forming function can be regulated by rationally controlling its expression level, thereby achieving nitrogen fixation efficiency and nodule-forming characteristics suitable for plant needs.

[0100] 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 that do not specify specific conditions are generally performed according to conventional conditions such as those described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Science Press, or according to the manufacturer's recommendations.

[0101] Experimental steps and specific implementation methods

[0102] 1. Plant materials and growing conditions

[0103] Ecotype R108 of Tribulus terrestris.

[0104] Medicago truncatula mutant nin-1: nin-1 deletion, see Marsh JF, Rakocevic A, Mitra RM, Brocard L, Sun J, Eschstruth A, Long SR, Schultze M, Ratet P, Oldroyd GE. 2007. Medicago truncatula NIN is essential for rhizobial-independent noduleorganogenesis induced by autoactive calcium / calmodulin-dependent proteinkinase. Plant Physiol 144, 324-335.

[0105] The Medicago truncatula mutant nlp1-1: nlp1-1 deletion, see Lin JS, Li X, Luo Z, Mysore KS, Wen J, Xie F. 2018. NIN interacts with NLPs to mediate nitrate inhibition of nodulation in Medicago truncatula. Nat Plants 4, 942-952.

[0106] Illuminated incubator: Plant cultures for detecting transcription levels are grown on plant culture medium FP.

[0107] Artificial climate chamber: Plants or tobacco plants with observed phenotypes were grown in soil containing vermiculite and perlite (1:1).

[0108] Temperature: 22℃ / 18℃ (day / night); Photoperiod: 16h / 8h (day / night); Irradiance: 250μmol·m -2 ·s -1 The relative humidity is 70%.

[0109] 2. Strains and their growth conditions

[0110] Escherichia coli: DH5α cultured in LB medium, solid cultured in a 37°C incubator, and liquid cultured in a 37°C shaker.

[0111] (1) Agrobacterium: Agrobacterium AR1193 (A. rhizogenes) was used for the root transformation experiment of alfalfa, and Agrobacterium GV3101 (A. tumefaciens) was used for the transient transformation experiment of tobacco. All Agrobacterium were cultured in LB medium, solid cultured in a constant temperature incubator at 28℃, and liquid cultured in a constant temperature shaker at 28℃.

[0112] (2) Rhizobia: Sinorhizobium meliloti 2011 / LacZ (Sm2011 is a wild-type rhizobia, in which lacZ is introduced) was used to inoculate alfalfa. The rhizobia were grown in TY medium, with solid culture in a 28°C incubator and liquid culture in a 28°C shaker.

[0113] Example 1: Cloning of genes and promoters

[0114] The amino acid sequence of the MtHHO1 protein (SEQ ID NO:1):

[0115] MVSMQFHHKMHSHKMEFRDYILALEEEKKKIQVFPRDLPLSLELVTQAIETCKQQLFGTQSECSEQTSTDEGLVFEEFIPIKKRALSPDCDENDEEDDDDEEQHSSHKMKSDWLRSVQLWNPNPSSAKEDVPRKTNVVEVKRNGGAFQPFHKEEIAAEKDNALESDKAPTSSPQVPATSSTEPVPESGSKKDDKGQRKQRRCWSQELHKRFLHALQQLGGSNSATPKQIRELMKVDGLTNDEVKSHLQKFRLHTRRSPIIHNNSNSHTAPMFLVGNIYVQPQEYAAVATKTTVSGELTTVTTPTGIYAPVATHPSSTTTVIKPKSKKFELSENSHSVERVVAHSNSPASSCSTHTPTTSRC*

[0116] Nucleotide sequence of the MtHHO1 gene (SEQ ID NO:2):

[0117]

[0118] MtHHO1 promoter sequence (SEQ ID NO:3):

[0119]

[0120] Using alfalfa (wild-type A17) DNA as a template, promoters and gene fragments were amplified by PCR under appropriate conditions using high-fidelity enzymes and primers (MAX, Vazyme; refer to the instruction manual for amplification system and procedure); bands of the target size were excised by gel electrophoresis, and the target fragments were recovered using a gel extraction kit.

[0121] Example 2: Preparation and Expression Analysis of Rhizobium Plants

[0122] 1. Construct promoter-fused GUS vectors and overexpression vectors

[0123] First, primers were designed, and attB homologous arms were added to both ends of the MtHHO1 promoter or gene primers, as shown in Table 1. Using alfalfa DNA as a template, the target fragment was mixed with the intermediate vector pDONR-207 in a certain ratio, Gateway BP recombinase was added, and the reaction was carried out overnight at 22°C. The reaction product was transformed by heat shock in a 42°C water bath, and 1 mL of antibiotic-free liquid LB was added after 30 seconds. After incubation on a shaker at 37°C for 50 minutes, the mixture was evenly spread onto LB solid medium containing gentamicin resistance and incubated upside down at 37°C overnight. The next day, positive clones were identified by PCR, and the culture was shaken and plasmids were extracted and sent for sequencing.

[0124] The correctly sequenced plasmid pDONR207-pHHO1 was mixed with the target vectors pKGWFS7-GUS-NLS-DsRed (Gateway vector library); pDONR207-gHHO1 (g:genome) and pUB-GW-GFP (Gateway vector library); and the target vectors pDONR207-HHO1-RNAi and pUB-GWS-GFP were mixed in appropriate proportions. After adding Gateway LR recombinase, the mixture was incubated overnight at 22°C. Then, heat shock transformation was performed, following the experimental procedures described above. Finally, the correctly sequenced plasmids pKGWFS7-pHHO1-GUS-NLS-DsRed, pUB-gHHO1-GFP, and pUB-HHO1-RNAi-GFP vectors were obtained.

[0125] The final vector was electroporated into Agrobacterium rhizogenes AR1193, antibiotic-free LB was added, and the mixture was incubated at 28°C for 1 h; it was then plated on LB plates with the corresponding antibiotic and incubated at 28°C for 48 h; colony PCR was performed using primers to verify the colony, and the correct clones were picked and cultured. The above components and the corresponding empty vectors were then transformed through the rhizogenes experiment.

[0126] Table 1

[0127]

[0128]

[0129] 2. Sterilization and germination of alfalfa seeds

[0130] Place the alfalfa seeds on sandpaper and gently rub them with your fingers to break the seed coat of each seed so that they can absorb water and swell. After grinding all the seeds, transfer them to clean 10ml centrifuge tubes. In a clean bench, add about 5ml of 10% sodium hypochlorite solution and invert the tubes thoroughly for 3-5 minutes.

[0131] Use a pipette to remove excess sodium hypochlorite liquid, add sterile water, rinse off any remaining sodium hypochlorite, wash 4-5 times, add sterile water again, and place on a shaker at 28°C to allow for full absorption and swelling.

[0132] The water can be changed multiple times during this period to remove impurities; the above process must be carried out in a sterile laminar flow hood to avoid seed contamination.

[0133] After the seeds have fully absorbed the water, remove excess moisture and spread them evenly on DWA (agar-only) solid culture medium. Incubate them in the dark and upside down in a refrigerator at 4°C for 3-5 days.

[0134] Then, remove the seeds from the refrigerator, place them upside down in a light incubator, and culture them in the dark for 12 hours. Once the hypocotyl has elongated to 3-5 cm, they can be used for root development or planted in greenhouse soil.

[0135] 3. Extraction of total RNA from plant roots and real-time quantitative PCR (RT-qPCR)

[0136] Roots of wild-type Alfalfa R108, mutant nin-1, and nlp1-1 were collected at different time points (12h, 1d, 3d, 5d, 7d, 10d, 14d, or 3h) after inoculation with rhizobia. 50-100 mg of each root was added to a steel ball and placed in liquid nitrogen. After cooling, the roots were quickly placed on a grinder clamp and ground at 60 Hz for 1 min, repeated 2-3 times. 800 μL of TRIzol Reagent (Invitrogen) was added, vortexed, and incubated on ice for 10 min. 200 μL of chloroform was added, mixed, and incubated on ice for 10 min, then centrifuged at 12000 rpm for 10 min. 300 μL of the supernatant was collected, and an equal volume of isopropanol was added. The mixture was incubated at room temperature for 10 min, then centrifuged at 12000 rpm for 10 min. The supernatant was discarded, and 1 mL of [unspecified substance] was added. Use 75% ethanol, 7500 rpm / 5 min, remove the supernatant, dry at room temperature, add 30-50 μL RNase-Free ddH2O, and let stand at 65℃ for 10 min; finally, measure the RNA concentration and adjust it to a suitable storage concentration for later use.

[0137] Using a reverse transcription kit One-Step gDNA Removal and cDNA SynthesisSuperMix were used to obtain the required cDNA. Primer sequences are shown in Table 2. The induction levels of HHO1 in roots of R108 and nin-1 at different time points (12h, 1d, 3d, 5d, 7d, 10d, 14d) after inoculation with rhizobium were detected by real-time quantitative PCR; or the induction level of HHO1 in roots of nlp1-1 3h after nitrate treatment.

[0138] Table 2

[0139]

[0140] The results showed that HHO1 was induced in R108 at 12 h, and the fold increase in induction increased at 5 days, reaching its peak at 14 days. However, in nin-1, the transcriptional level of HHO1 was suppressed. Figure 1 A). This indicates that HHO1 is induced by rhizobia and depends on the symbiotic-specific transcription factor NIN.

[0141] Compared to R108, HHO1 expression in the nlp1-1 mutant is almost unaffected by nitrate-induced expression. Figure 1 B) indicates that HHO1 is nitrate-induced to express the transcription factor NLP1.

[0142] 4. Rooting transformation of alfalfa

[0143] Agrobacterium AR1193 constructed from pKGWFS7-pHHO1-GUS-NLS-DsRed or pUB-gHHO1-GFP was activated on LB solid medium containing the corresponding antibiotic. Single colonies were picked and transferred to LB liquid medium containing the corresponding antibiotic and incubated overnight at 28°C with shaking. 100 μL of the bacterial suspension was transferred to LB liquid medium containing the corresponding antibiotic at a ratio of 1:50 and incubated overnight at 28°C with shaking. Meanwhile, the imbibed alfalfa seeds were placed in an inverted light incubator for germination for about 14 hours. On the second day, the bacterial cells were enriched and resuspended in fresh antibiotic-free LB medium for later use. Germinated seedlings were removed and placed in Petri dishes containing sterile water. The seed coat was removed with forceps, and the hypocotyl of the alfalfa cotyledons (1-1.5 cm to the root tip) was completely removed. The seedlings were thoroughly immersed in the resuspended Agrobacterium for 10-20 minutes and then placed in an MFP plant culture medium for about one week. The petri dishes were placed at an angle in a 22°C light incubator for cultivation. After one week, the non-transformed roots at the hypocotyl of the seedlings were removed. The seedlings were placed between two layers of filter paper and transferred to HRE medium containing termethin. After two weeks of growth, the seedlings were removed and placed under a fluorescence stereomicroscope to remove the non-transformed roots. Finally, the rooted seedlings were planted in nitrogen-free soil in a greenhouse and allowed to grow until they were inoculated with rhizobia.

[0144] 5. Rhizobium inoculation

[0145] Plants that were transformed with pKGWFS7-pHHO1-GUS-NLS-DsRed or pUB-gHHO1-GFP were planted in nitrogen-free soil in a greenhouse and allowed to grow for one week before being inoculated with rhizobia. Meanwhile, the frozen Sm2011 / LacZ strain was taken out from the -80℃ ultra-low temperature freezer and spread on TY solid medium containing streptomycin resistance. It was incubated upside down in a 28℃ incubator for 2 days. The activated bacterial cells were picked and transferred to 3-5 mL of TY liquid medium containing streptomycin resistance and cultured on a shaker at 28℃ for 12-18 h. The culture was then transferred to 50-100 mL of new TY liquid medium at a ratio of 1:50 and cultured at 28℃ until the OD600 reached about 1.0, about 12-18 h. The bacterial culture was aliquoted into 50 mL centrifuge tubes and centrifuged at 4000 rpm for 15 min at 4℃. The supernatant was discarded, and an appropriate amount of sterile water was added to gently resuspend the bacterial cells. The bacterial cells were then diluted with sterile water to an OD600 of 0.02-0.03. Finally, the transformed plants were inoculated using an inoculation bottle.

[0146] Example 3: GUS staining to observe the expression of the HHO1 promoter

[0147] Plants with roots transformed into pKGWFS7-pHHO1-GUS-NLS-DsRed were inoculated with Sm2011 / LacZ. At selected time points (5 days, 7 days, 10 days, and 14 days), the plants were pulled up, washed, and the non-transformed roots were removed. The transformed roots were collected into 5 mL EP tubes and added to GUS staining solution. The tubes were then incubated at 37°C and the staining was observed every 20 minutes.

[0148] Table 3

[0149] GUS staining solution (pH 7.0) 200L <![CDATA[NaH2PO4·2H2O]]> 1.17g <![CDATA[Na2HPO4·12H2O]]> 4.47g <![CDATA[K3[Fe(CN)]6]]> 0.066g <![CDATA[K4[Fe(CN)]6·3H2O]]> 0.084g 0.5M EDTA 2mL Triton X-100 0.2mL 25g / ml X-Gluc (in DMF) 4mL

[0150] The results showed that HHO1 was expressed not only in root hair cells and cortical cells, but also in the meristematic zone of young root nodules, mature root nodules, and nitrogen-fixing cells near the meristematic zone. Figure 1 C).

[0151] On the other hand, after treatment with 5 mL KCl / KNO3 for 30 min, it was found that under nitrate treatment, HHO1 was expressed in root hairs, root epidermis, cortex, and pericycle cells. Figure 1 D).

[0152] Example 4: Nodular phenotype with HHO1 overexpression: observation of infection events

[0153] Plants with rootlets transformed into pUB-gHHO1-GFP were inoculated with Sm2011 / LacZ for 6 days. The plants were then removed, washed, and non-transformed roots were removed. The transformed roots were collected in clean EP tubes, submerged in fixative, and vacuumed once (10-15 min). The tubes were then fixed at 28°C for 2 hours. The fixative was then washed off twice with phosphate buffer, 15 min apart. After washing, excess liquid was aspirated, and a certain amount of X-Gal staining solution was added. The EP tubes were inverted to ensure root submersion, and the tubes were treated in a black box at room temperature for 24-36 hours. The staining solution was washed off based on the final staining. After staining, the tubes were washed three times with phosphate buffer, 10 min apart, followed by two 15 min washes with sterile water. Then, they were washed twice with 10% NaClO, and finally twice with sterile water. The tubes were then placed in phosphate buffer and the infection phenotype was observed under a confocal laser microscope.

[0154] Table 4

[0155] 0.1M phosphate buffer (pH 7.0) 1L <![CDATA[KH2PO4]]> 5.3g <![CDATA[K2HPO4]]> 13.9g

[0156] Table 5

[0157] Fixative 50mL 10% glutaraldehyde 2.5mL 0.1M phosphate buffer 47.5mL

[0158] Table 6

[0159] X-Gal staining solution <![CDATA[100mM K4Fe(CN)6]]> 200ul <![CDATA[100mM K3Fe(CN)6]]> 200ul 2% X-Gal in DMF 150ul 0.1M phosphate buffer 3.2mL

[0160] Statistical results showed that, compared with the empty vector, the number of infection sites (Foci), infection lines (IT), and root nodule primordia (NP) in HHO1-overexpressing plants were significantly reduced. Figure 2 B).

[0161] Example 5: Nodulation phenotype with HHO1 overexpression: Observation of nodulation events

[0162] Plants with roots transformed with pUB-gHHO1-GFP were inoculated with rhizobium Sm2011 / LacZ for 2 weeks, then the plants were removed and washed.

[0163] Under a fluorescence microscope, after removing non-transformed roots, the nodulation phenotype of HHO1-overexpressing plants was observed and statistically analyzed.

[0164] Statistical results showed that, compared with the empty vector, the number of root nodules in plants overexpressing HHO1 was significantly reduced. Figure 2 (A and 2C).

[0165] Example 6: Nodular phenotype with HHO1 overexpression: Detection of nitrogenase activity

[0166] After observing and statistically analyzing the nodulation phenotype of HHO1-overexpressing plants, root nodules were collected for nitrogenase activity detection. The specific procedure was as follows: all root nodules from three plants were placed in 10mL sealed brown glass bottles, with 5 samples from 15 plants in each experimental group. One mL of air was first extracted from the glass bottle using a syringe, followed by the injection of 1 mL of acetylene gas into the glass tube. The reaction was carried out at 28℃ in the dark for 2 hours, then terminated with 1 mL of 1M NaOH (ensuring all root nodules were submerged in NaOH), and the reaction was awaited for measurement. The gas chromatograph was preheated beforehand. A 0.25mL sample of gas from the glass bottle was injected into the gas chromatograph using a syringe, and the acetylene and ethylene peak values ​​were recorded to calculate nitrogenase activity.

[0167] The results showed that HHO1 overexpression significantly reduced nitrogenase activity (ARA, Acetylene Reduction Activity). Figure 2 D).

[0168] Example 7: Nodulation phenotype of HHO1RNAi: Observation of nodulation events

[0169] Plants with roots transformed with pUB-HHO1-RNAi were inoculated with rhizobium Sm2011 / LacZ for 2 weeks, then the plants were removed and washed.

[0170] 1. Expression analysis of HHO1

[0171] The transcriptional level of HHO1 in the roots of HHO1 RNAi plants was detected by real-time quantitative PCR.

[0172] The results showed that, compared with the empty vector, HHO1 levels in the roots of HHO1 RNAi plants decreased by 50%. Figure 3 C).

[0173] 2. Phenotypic Analysis of Nodules

[0174] Under a fluorescence microscope, after removing non-transformed roots, the nodulation phenotype (average number of root nodules) of HHO1 RNAi plants was observed and statistically analyzed.

[0175] Statistical results showed that, compared with the empty vector, HHO1 overexpression resulted in an increased number of root nodules in the plants. Figure 3 (A and 3B).

[0176] Example 8: Screening for substances used to regulate traits

[0177] Test subject: Alfalfa, which normally expresses endogenous HHO1.

[0178] Test group: The above test subjects were treated with the candidate substances;

[0179] Control group: No candidate substance was introduced.

[0180] The expression and activity of HHO1 in the test group and the control group of alfalfa were detected and compared.

[0181] If the expression or activity of HHO1 in the test group is statistically higher (at least 50% higher) than that in the control group, it indicates that the candidate is a potential substance for reducing rhizobium infection (reducing infection events), reducing the number of root nodules in plants, and reducing nitrogenase activity.

[0182] If the expression or activity of HHO1 in the test group is statistically lower (at least 50% lower) than that in the control group, it indicates that the candidate is a potential substance that can be used to increase the number of root nodules in plants.

[0183] 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. An application of HHO1 for regulating nitrogen fixation traits in legumes, or for use as a regulatory target in the preparation of regulatory molecules that regulate nitrogen fixation traits in legumes; said nitrogen fixation traits include: Characteristics of root nodule number, rhizobium infection, and nitrogenase activity.

2. The use as described in claim 1, characterized in that, The regulatory molecule is an HHO1 upregulated molecule, which reduces rhizobium infection, reduces the number of root nodules in plants, and lowers nitrogenase activity; preferably, the upregulated molecule includes: an exogenous HHO1 encoding gene or an expression construct or vector containing the encoding gene.

3. The use as described in claim 1, characterized in that, The regulatory molecule mentioned is an HHO1 downregulatory molecule, which increases the number of root nodules in plants; Preferably, the downregulating molecule includes: a reagent for loss-of-function mutation of HHO1, a reagent for knocking out or silencing HHO1, or a reagent for inhibiting HHO1 activity; preferably, it includes: an interfering molecule that specifically interferes with the expression of the gene encoding HHO1, a CRISPR gene editing reagent, a homologous recombination reagent, or a site-directed mutagenesis reagent targeting HHO1; preferably, the downregulating molecule is an interfering molecule that includes RNAi formed by primers (e.g., annealing to form a double strand) of the nucleotide sequences shown in SEQ ID NO:8 and SEQ ID NO:

9.

4. The use as described in any one of claims 1-3, characterized in that, The amino acid sequence of the HHO1 polypeptide is selected from the following group: (i) A polypeptide having the amino acid sequence shown in SEQ ID NO:1; (ii) A polypeptide with ≥80% homology to the amino acid sequence shown in SEQ ID NO:1 and having the regulatory trait function; (iii) A polypeptide 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:1; or, (iv) A polypeptide formed by adding a tag sequence or restriction enzyme site sequence to the N or C end of the polypeptide with the amino acid sequence shown in SEQ ID NO:1, or by adding a signal peptide sequence to its N end.

5. A method for regulating nitrogen-fixing traits in legumes, comprising: Regulates the expression or activity of HHO1 in legumes; The nitrogen-fixing traits include: the number of root nodules, rhizobium infection, and nitrogenase activity.

6. The method as described in claim 5, characterized in that, The method aims to increase the expression or activity of NHHO1 in plants, or to increase the expression or activity of HHO1 in plants with low HHO1 expression, thereby reducing rhizobium infection, reducing the number of root nodules, and decreasing nitrogenase activity. The method includes: introducing the HHO1 coding gene or an expression construct or vector containing the coding gene into plants; performing gain-of-function mutations on HHO1; promoting HHO1 expression by expressing an enhancing promoter or a tissue-specific promoter; or promoting HHO1 expression by an enhancer.

7. The method as described in claim 6, characterized in that, Downregulating the expression or activity of HHO1 in plants, thereby increasing the number of root nodules in plants; the method includes: performing a loss-of-function mutation on HHO1 in plants containing HHO1, knocking out or silencing the gene encoding HHO1, or inhibiting the activity of HHO1. Preferably, the loss-of-function mutation of HHO1 includes: targeted modification of the coding gene of HHO1 to reduce its function / activity; more preferably, causing a frameshift in its coding gene to terminate translation prematurely. Preferably, the expression of the HHO1 coding gene is interfered with by an interfering molecule; more preferably, the interfering molecule comprises RNAi formed by primers of the nucleotide sequences shown in SEQ ID NO:8 and SEQ ID NO:

9.

8. The method as described in any one of claims 5-7, characterized in that, After preparing leguminous plants with altered nitrogen-fixing traits using the method described above, the method further includes: hybridizing the plants whose HHO1 expression or activity has been regulated with plants that have not introduced the HHO1 polypeptide or its encoding gene to obtain hybrid offspring.

9. The method as described in any one of claims 1-4 or the use as described in any one of claims 5-8, characterized in that, The legumes mentioned include: alfalfa, soybean, birdsfoot root, pea, peanut, kidney bean, mung bean, red bean, broad bean, cowpea, or milkvetch.

10. The use of HHO1, a legume, as a molecular marker for identifying nitrogen-fixing traits in legumes, or as a molecular marker for targeted plant screening; wherein, The nitrogen-fixing traits include: the number of root nodules, rhizobium infection, and nitrogenase activity. Preferably, the expression or sequence characteristics of HHO1 protein or its gene in the test plant are identified; if the HHO1 protein or its gene is highly expressed or highly active in the test plant, it is a plant with reduced rhizobium infection, fewer root nodules, and lower nitrogenase activity; if the HHO1 protein or its gene is low expressed or low active in the test plant, it is a plant with a high number of root nodules.

11. A legume cell, tissue, or organ containing an exogenous regulatory molecule of HHO1; in, The regulatory molecule is an HHO1 upregulatory molecule, including: an exogenous HHO1 encoding gene or an expression construct or vector containing the encoding gene; or, the regulatory molecule is an HHO1 downregulatory molecule, including: a reagent for loss-of-function mutation of HHO1, a reagent for knocking out or silencing HHO1, a reagent for inhibiting HHO1 activity; preferably including: an interfering molecule that specifically interferes with the expression of the HHO1 encoding gene; a CRISPR gene editing reagent, a homologous recombination reagent, or a site-directed mutagenesis reagent targeting HHO1.

12. A method for screening substances that regulate nitrogen-fixing traits in legumes, comprising: (1) The candidate substance is added to a test system containing HHO1; (2) Detect the system and observe the expression or activity of HHO1. If the expression or activity of HHO1 is increased, it indicates that the candidate substance is a substance that can be used to reduce rhizobium infection, reduce the number of root nodules in plants, and reduce nitrogenase activity. If the expression or activity of HHO1 is reduced, it indicates that the candidate substance can be used to increase the number of root nodules in plants.

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