Application of cell wall protein in regulation and control of nitrogen fixation efficiency of plant root nodules

Abstract

The invention discloses application of cell wall protein to regulation and control of nitrogen fixation efficiency of plant root nodules. The invention discloses the important function of the cell wall protein in regulating and controlling the nitrogen fixation of the plant root nodule for the first time, particularly clarifies the action mechanism of the GmLRX3 and the GmLRX4 in regulating and controlling the proliferation of the bacteria-like body, and lays a foundation for deeply exploring the regulation and control mechanism of the NCRs-deleted leguminous plant symbionts in the future.

Description

Technical Field

[0001] The present application relates to the field of plant genetic engineering, and in particular to the use of cell wall proteins in regulating the nitrogen fixation efficiency of plant nodules. Background Art

[0002] Legumes can effectively convert atmospheric nitrogen into a nitrogen source that can be used by plants through a symbiotic relationship with rhizobia, a process called symbiotic nitrogen fixation. However, the control of rhizobia proliferation within the symbiont is essential for maintaining the energy balance of the plant. Studies have shown that some legumes inhibit the excessive proliferation of rhizobia by secreting nodule-specific cysteine-rich peptides (NCRs), thereby promoting their transformation into bacteroids with nitrogen-fixing ability. For example, alfalfa is known to use NCRs to promote the differentiation of rhizobia into bacteroids, causing them to lose their reproductive ability, thereby controlling the number of rhizobia (Velde et al., 2010; Wang et al., 2010).

[0003] Leguminosae plants form nodule structures through symbiosis with rhizobia, thereby obtaining nitrogen sources in the nitrogen fixation reaction. However, the uncontrolled number of rhizobia will cause excessive energy consumption for the plant and hinder the efficiency of the nitrogen fixation process. Therefore, leguminous plants need effective mechanisms to control the proliferation of rhizobia in the symbiosis. Previous studies have shown that some leguminous plants, such as the inverted repeat-lacking clade (IRLC) and Aeschynomenespecies, can control the formation of bacteroids and the proliferation of rhizobia by secreting NCRs. However, the mechanism by which leguminous plants lacking NCRs (such as soybean and Lotus japonicus) regulate the proliferation of rhizobia is still unclear.

[0004] Although NCRs are known to regulate the proliferation of rhizobia in some legumes, the mechanism of regulating the proliferation of bacteroids in legumes lacking NCRs (such as soybean and Lotus japonicus) remains unclear, which limits the in-depth application of the symbiotic nitrogen fixation mechanism of legumes in this field. Summary of the invention

[0005] Based on this, it is necessary to provide at least one use of a cell wall protein in regulating the nitrogen fixation efficiency of plant nodules.

[0006] In a first aspect of the present application, there is provided use of a recombinant expression vector comprising a nucleic acid molecule encoding a cell wall protein in regulating the number of rhizobia in a plant.

[0007] In some embodiments, the recombinant expression vector regulates the nitrogen fixation efficiency of plant nodules by regulating the number of rhizobia in the plant.

[0008] In some embodiments, the cell wall protein includes at least one of GmLRX3 and GmLRX4.

[0009] In a second aspect of the present application, a method for cultivating an efficient nodulation and nitrogen-fixing plant is provided, the method comprising increasing the content and / or activity of a nucleic acid molecule encoding a cell wall protein in a target plant, thereby effectively controlling the number of rhizobia and obtaining a plant with efficient nitrogen fixation. For example, a plant having a different number of rhizobia from the target plant is obtained.

[0010] In some embodiments, the method comprises changing the content and / or activity of a nucleic acid molecule encoding a cell wall protein in a target plant, thereby effectively controlling the number of rhizobia and obtaining a plant with efficient nitrogen fixation.

[0011] In some embodiments, the method comprises increasing the expression of a nucleic acid molecule encoding a cell wall protein in a plant of interest.

[0012] In some embodiments, the expression level is increased by at least one of the following methods:

[0013] 1) increasing the copy number of the nucleic acid molecule;

[0014] 2) expressing the nucleic acid molecule under the drive of a strong promoter;

[0015] 3) increasing the regulatory element of the nucleic acid molecule to overexpress it, wherein the regulatory element is selected from the group consisting of an enhancer element, an element that improves mRNA stability, an element that enhances translation efficiency, and an element that enhances protein secretion;

[0016] 4) Increase the ribosome binding sites of the nucleic acid molecule.

[0017] In some embodiments, increasing the expression level is achieved by introducing the nucleic acid molecule into the target plant.

[0018] In some embodiments, a plant of interest is transfected with a recombinant expression vector comprising a nucleic acid molecule encoding a cell wall protein.

[0019] In some embodiments, the nucleic acid molecule is introduced into the target plant by hairy root transformation.

[0020] In some embodiments, a target plant with a target gene knocked out is obtained by stable transformation.

[0021] In some embodiments, the nucleic acid molecule is introduced into an underground portion of a plant of interest.

[0022] In some embodiments, the nucleic acid molecule is introduced into the peribacteroid space (PBS) of the plant of interest.

[0023] In some embodiments, the plant is selected from the group consisting of Fabales, Cucurbitales, Rosales, and Fagales.

[0024] This application revealed for the first time the important role of cell wall proteins in this regulatory mechanism by screening host proteins rich in symbiont membranes and perimycin cavities in soybeans. Through a series of mutant analyses and phenotypic studies, the action mechanism of cell wall proteins represented by GmLRX3 and GmLRX4 in regulating bacteroid proliferation was further clarified, laying the foundation for in-depth exploration of the regulatory mechanism of NCRs-deficient legume symbionts in the future. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the technical solutions in the implementation methods and examples of the present application and to more completely understand the present application and its beneficial effects, the following is a brief introduction to the drawings required for use in the description of the implementation methods or examples. Obviously, the drawings described below are only some embodiments of the present application. For those skilled in the art, other drawings can be obtained based on these drawings without creative work. It should also be noted that the drawings are all drawn in a simplified form and are only used to conveniently and clearly assist in explaining the present application.

[0026] Figure 1 It is the expression level of GmLRX4 in one embodiment of the present application.

[0027] Figure 2 This is the subcellular localization of GmLRX4 in one embodiment of the present application.

[0028] Figure 3 This is a phylogenetic analysis of the soybean and Arabidopsis LRX families in one embodiment of the present application.

[0029] Figure 4 It is the expression level of the GmLRX4 homologous gene in soybean in one embodiment of the present application.

[0030] Figure 5 The target sequence and editing status of the lrxs-related mutants used in one embodiment of the present application.

[0031] Figure 6 This is the phenotype of Gmlrx3 / 4 and Ljlrx4 mutants in one embodiment of the present application.

[0032] Figure 7 It is the expression level of cell division related genes in rhizobia in one embodiment of the present application. DETAILED DESCRIPTION

[0033] In order to facilitate the understanding of the present application, the present application will be described more fully below with reference to the relevant drawings. The preferred embodiments of the present application are given in the drawings. However, the present application can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present application more thorough and comprehensive.

[0034] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application belongs. The terms used herein in the specification of this application are only for the purpose of describing specific embodiments and are not intended to limit this application. The term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0035] In this application, unless otherwise specified, "one or more" means any one of the listed items or any combination of the listed items. Similarly, "one or more" and the like, when used in other ways to mean "one or more", are also understood in the same way unless otherwise specified.

[0036] The terms "combination thereof", "any combination thereof", "any combination thereof" and the like used in this application include all suitable combinations of any two or more of the listed items.

[0037] In this application, the word "suitable" in "suitable combination", "suitable method", "any suitable method", etc. is based on the ability to implement the technical solution of this application, solve the technical problems of this application, and achieve the expected technical effects of this application.

[0038] In this application, "further", "further", "particularly", "for example", "such as", "example", "for example", etc. are used for descriptive purposes, indicating that the previous and subsequent different technical solutions are related in terms of the content covered, but should not be understood as a limitation on the previous technical solution, nor can it be understood as a limitation on the protection scope of this application. In this application, unless otherwise specified, A (such as B) means that B is a non-limiting example of A, and it can be understood that A is not limited to B.

[0039] In this application, "optionally", "optional", and "optional" mean optional, that is, any one of the two parallel solutions of "yes" or "no". If there are multiple "optional" in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "optional" is independent. Unless otherwise specified, the descriptions of "optionally include", "optionally contain", etc. in this application, taking "optionally include" as an example, mean "may include or not include".

[0040] The terms "contain", "include" and "include" used in this application are synonymous, which are inclusive or open-ended and do not exclude additional, uncited members or features. Members or features include materials or components, etc.; non-limiting examples of members or features include actions, conditions for the occurrence of actions, timing, states, etc.

[0041] In this application, the technical features or technical solutions described in an open language include closed technical features or technical solutions composed of the listed contents, and also include open technical features or technical solutions containing the listed contents.

[0042] In the present application, exemplary descriptions such as "in some implementation modes (or examples)" and "in one implementation mode (or example)" may include but are not limited to the following meanings: these solutions may be combined with other solutions in a suitable manner to form new technical solutions.

[0043] In the present application, in "the first aspect", "the second aspect", "the third aspect", "the fourth aspect", etc., the terms "first", "second", "third", "fourth", etc. are used only for descriptive purposes and cannot be understood as indicating or implying relative importance or quantity, nor can they be understood as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first", "second", "third", "fourth", etc. only serve the purpose of non-exhaustive enumeration and description, and it should be understood that they do not constitute a closed limitation on quantity.

[0044] In this application, unless otherwise specified, the term "bacteroid" in this application refers to the fact that leguminous plants can interact with soil rhizobia to form nodules and perform symbiotic nitrogen fixation. After rhizobia are released into the cytoplasm of root cells, they proliferate and differentiate into a special state with the ability to fix nitrogen.

[0045] In this application, unless otherwise specified, the term "symbiont" in this application refers to a structure formed by one or more bacteriomorphs surrounded by a symbiont membrane derived from a plant membrane.

[0046] In this application, unless otherwise specified, the term "nucleic acid" in this application has the well-known meaning in the technical field, also known as "polynucleotide", which is a molecule formed by multiple nucleotide monomers.

[0047] In this application, unless otherwise specified, "gene" has the well-known meaning in the art, and refers to a polynucleotide fragment that carries the genetic information of an organism, which can be a coding gene or a regulatory gene. "Coding gene" refers to a polynucleotide fragment that can synthesize a protein or its fragment when expressed, and is an effective gene fragment that encodes a protein or its fragment. A gene includes a coding region and a non-coding region. The coding region is a region that can be transcribed into the corresponding mRNA and then guide protein synthesis, that is, a region that can encode a protein. The non-coding region is a region that cannot be transcribed into the corresponding mRNA. The coding gene includes a coding sequence. "Coding sequence" refers to a nucleotide sequence that completely corresponds to the codon of a protein, and the sequence does not contain nucleotide units that do not correspond to the codon of the protein.

[0048] In this application, unless otherwise specified, "protein" and "protein" have the same meaning, both correspond to "protein", and can be used interchangeably. "Protein", "polyamino acid", "peptide" and "polypeptide" have the same meaning, can be used interchangeably, and all include at least two amino acid units.

[0049] In this application, unless otherwise specified, the term "vector" refers to a nucleic acid carrier into which a polynucleotide can be inserted. When a vector can express a protein encoded by the inserted polynucleotide, the vector is called an expression vector. The vector can be introduced into a host cell (such as a plant cell) by transformation, transduction or transfection, so that the genetic material elements it carries are expressed in the host cell. The vector can be a plasmid.

[0050] In some embodiments, a "vector" is a nucleic acid molecule carrying a gene of interest, which can deliver the gene of interest in a desired manner, for example, can deliver the gene of interest into a host cell.

[0051] In the present application, unless otherwise specified, the term "expression cassette" refers to a nucleic acid sequence that at least includes a promoter and a target gene, which can express the target gene in a specific cell to synthesize the protein encoded by the target gene.

[0052] In this application, unless otherwise specified, the term "strong promoter" refers to a promoter that has a high affinity for RNA polymerase and can guide the synthesis of a large amount of mRNA. Exemplary strong promoters include lacP, trpP, tacP, λPL, alcohol dehydrogenase promoter, CaMV promoter, polyhedrin gene promoter, SV40 promoter, adenovirus late gene promoter, etc.

[0053] In the present application, unless otherwise specified, the term "cell wall protein" refers to a class of LRX proteins comprising an extensin domain capable of binding to cell wall polysaccharides and an LRR domain capable of interacting with other proteins.

[0054] In the present application, when it comes to a numerical interval (i.e., a numerical range), unless otherwise specified, the distribution of the optional numerical values ​​in the numerical interval is considered to be continuous, and includes the two numerical endpoints (i.e., the minimum and maximum values) of the numerical interval, and each numerical value between the two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to an integer in the numerical interval, it includes the two endpoint integers of the numerical range, and each integer between the two endpoints, which is equivalent to directly listing each integer. When multiple numerical ranges are provided to describe features or characteristics, these numerical ranges can be merged. In other words, unless otherwise specified, the numerical range disclosed herein should be understood to include any and all sub-ranges included therein. The "numerical value" in the numerical interval can be any quantitative value, such as a number, a percentage, a ratio, etc. "Numerical interval" allows broadly including numerical interval types such as percentage intervals, ratio intervals, and ratio intervals.

[0055] In this application, if there are multiple steps involved in the method flow, unless there is a clear different description in this document, there is no strict order restriction for the execution of these steps, and they can be executed in other orders than described. Moreover, any step can include multiple sub-steps or multiple stages, and these sub-steps or stages are not necessarily executed at the same time, but can be executed at different times, and their execution order is not necessarily sequential, but can be executed in turn, alternating or simultaneously with other steps or parts of sub-steps or stages of other steps.

[0056] The present application relates to the screening of host factors that control the proliferation of bacteroids in the symbionts of legumes lacking NCRs, aiming to reveal the regulatory mechanism of host factors on the proliferation of bacteroids. By separating and analyzing the proteins in the soybean symbiont membrane and the perimycin cavity, it was found that the high expression of the rich cell wall protein GmLRX4 and its homologous gene GmLRX3 in nodules was closely related to the proliferation of bacteroids. By constructing Gmlrx3 / 4 double mutant plants, it was proved that the loss of GmLRX3 and GmLRX4 can significantly increase the number of bacteroids in the symbiont, indicating that this may be a conservative mechanism in legumes lacking NCRs. The present application provides a new perspective for a deeper understanding of the regulatory mechanism of bacteroid proliferation and lays a foundation for the research and application of the legume-rhizobium symbiotic system.

[0057] Specifically, the contribution of this application is at least:

[0058] It was found that GmLRX4 is localized in the perimycotic cavity: GmLRX4 protein is mainly localized in the perimycotic cavity of root nodule symbionts;

[0059] It is clear that the loss of GmLRX3 and GmLRX4 increases the number of bacteroids: By constructing double mutants of GmLRX3 and GmLRX4 and performing ultrathin sectioning and transmission electron microscopy analysis on the mutants, it was found that the number of bacteroids in the nodules of the double mutants increased significantly, indicating that GmLRX3 and GmLRX4 play an important role in regulating the proliferation of bacteroids;

[0060] Clarifying the phenotypic impact of the variants: The Gmlrx3 / 4 double mutant not only showed significant changes in bacteroid proliferation, but also revealed changes in rhizobium gene expression: by transcriptome sequencing of rhizobia in nodules of wild-type and double mutants, it was found that the expression of rhizobia cell division-related genes in the double mutant was significantly upregulated, indicating that the loss of GmLRX3 and GmLRX4 led to an increase in the number of bacteroids, which may be due to more active rhizobium division.

[0061] Based on the above findings, this application provides the following technical solutions.

[0062] One aspect of the present application provides the application of biomaterials related to cell wall proteins, which applications include but are not limited to: regulating the formation and / or growth of plant nodules, regulating the number of plant nodules, regulating the nitrogen fixation efficiency of plant nodules, cultivating plants with increased or decreased number of rhizobia in the plant body, and cultivating efficient nodulation and nitrogen fixation plants.

[0063] The biological materials in this application include but are not limited to:

[0064] a nucleic acid molecule encoding the cell wall protein;

[0065] an expression cassette comprising the nucleic acid molecule;

[0066] A recombinant expression vector comprising the nucleic acid molecule and / or expression cassette;

[0067] A recombinant microorganism comprising one or more of the nucleic acid molecule, the expression cassette and the recombinant expression vector;

[0068] A recombinant host cell comprising one or more of the nucleic acid molecule, the expression cassette and the recombinant expression vector;

[0069] A genetic plant tissue comprising one or more of the nucleic acid molecule, the expression cassette and the recombinant expression vector; and

[0070] A transgenic plant organ comprising one or more of the nucleic acid molecule, the expression cassette and the recombinant expression vector.

[0071] In some embodiments, the invention relates to the use of a recombinant expression vector comprising a nucleic acid molecule encoding a cell wall protein in regulating the nitrogen fixation efficiency of plant nodules.

[0072] In some embodiments, the recombinant expression vector regulates the nitrogen fixation efficiency of plant nodules by regulating plant nodule formation and / or growth.

[0073] Illustratively, in some embodiments, a recombinant expression vector comprising a gene encoding a cell wall protein is introduced into a plant cell to promote the formation and / or growth of nodules, thereby regulating the nitrogen fixation efficiency of the plant nodules.

[0074] The cell wall protein in the present application may be a conventional cell wall protein in the art. In some embodiments, the cell wall protein includes one or more of GmLRX3 and GmLRX4.

[0075] The present application also provides a method for cultivating efficient nodulation and nitrogen-fixing plants, which comprises changing the content and / or activity of the nucleic acid molecule encoding the cell wall protein as described above in the target plant, thereby effectively controlling the number of rhizobia and obtaining efficient nitrogen-fixing plants.

[0076] In some embodiments, the method comprises increasing the expression of a nucleic acid molecule encoding a cell wall protein in a plant of interest.

[0077] Methods for increasing expression levels may be conventional in the art, such as increasing the copy number of nucleic acid molecules, driving expression through a strong promoter, using regulatory elements, and adding ribosome binding sites, etc. The listed methods may be used alone or in combination.

[0078] In some embodiments, the expression level is increased by increasing the copy number of the nucleic acid molecule.

[0079] In some embodiments, the expression level of the nucleic acid molecule is increased by placing the nucleic acid molecule under the drive of a strong promoter.

[0080] In some embodiments, the regulatory element of the nucleic acid molecule is added to enable its overexpression, and the regulatory element is selected from the group consisting of an enhancer element, an element that increases mRNA stability, an element that enhances translation efficiency, and an element that enhances protein secretion.

[0081] In some embodiments, the expression level of the nucleic acid molecule is increased by increasing the ribosome binding site of the nucleic acid molecule.

[0082] In some embodiments, the increase in expression level is achieved by introducing the nucleic acid molecule into the target plant.

[0083] The nucleic acid molecules can be introduced into the target plant using conventional methods in the art. In some embodiments, the target plant is transfected with a recombinant expression vector containing a nucleic acid molecule encoding a cell wall protein.

[0084] In some embodiments, the nucleic acid molecule is introduced into the target plant by hairy root transformation.

[0085] In some embodiments, a target plant with a target gene knocked out is obtained by stable transformation.

[0086] The nucleic acid molecule can be introduced into any part of the target plant.

[0087] In some embodiments, the nucleic acid molecule is introduced into an underground portion of a plant of interest.

[0088] In some embodiments, the nucleic acid molecule is introduced into the perimyossary cavity of a plant of interest.

[0089] The plant in the present application can be a conventional plant in the art. Exemplarily, the plant is selected from the group consisting of Fabales, Cucurbitales, Rosales and Fagales.

[0090] In some embodiments, the plant may be a plant of the order Fabales, Cucurbitales, Rosales, or Fagales.

[0091] The present application also provides a method for reducing the number of rhizobia in a plant, the method comprising constructing a plant lacking a cell wall protein. The method for constructing a plant lacking a cell wall protein may be, for example, CRISPR / Cas9 technology.

[0092] In some embodiments, the cell wall protein is GmLRX4.

[0093] In other embodiments, the cell wall protein is GmLRX3.

[0094] In some embodiments, plants lacking GmLRX3 and GmLRX4 are constructed, for example, by deleting 26 bp and 5 bp in the CDS region, respectively, to cause premature termination of translation.

[0095] Some examples are provided below.

[0096] The embodiments of the present application will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods for which the conditions are not specified in the following examples are preferably referred to the guidance given in the present application, and can also be based on the experimental manual or normal conditions in the art, or can also be based on the conditions recommended by the manufacturer, or with reference to experimental methods known in the art.

[0097] Example 1

[0098] 1. Screening of host factors controlling bacteroid proliferation in NCRs-deficient legume symbioses

[0099] In order to find host factors that control the proliferation of bacteroids in legume symbionts lacking NCRs, the laboratory previously isolated and analyzed proteins in the soybean symbiont membrane and peribacterial cavity (PBS) (Luo et al., 2022). Although there is no complete cell wall structure in the symbiont (Brewin, 2004), the cell wall protein LEUCINE-RICH REPEATEXTENSIN 4 (GmLRX4) was found to be abundant in the symbiont membrane and PBS, especially in the PBS ( Figure 1 In addition, quantitative PCR (qRT-PCR) analysis showed that the expression level of GmLRX4 in nodules was higher than that in roots ( Figure 1 (B) These results suggest that GmLRX4 may play a role in symbiosis.

[0100] 2. GmLRX4 is localized in the perimicrobial cavity

[0101] In order to clarify the localization of GmLRX4 in PBS, a fusion GFP vector driven by the nodule-specific promoter LBc1 (Leghemoglobin) was constructed and transformed into soybeans by hairy root transformation. The positive nodules were cut into slices no larger than 50 µm and observed under a laser confocal microscope. The results showed that GmLRX4 formed a hollow structure in the infected cells, and the rhizobia were well embedded in it ( Figure 2 A), and the GmLRX4-labeled structure is encapsulated by the symbiont membrane ( Figure 2 These results indicate that GmLRX4 is localized in the PBS.

[0102] 3. Analysis of lrx Mutant Phenotypes

[0103] Soybean is a paleotetraploid with a high degree of gene redundancy, and the LRXs family members in Arabidopsis show obvious functional redundancy. Therefore, in order to clarify the function of GmLRX4, the homologous genes of GmLRX4 in soybean were first analyzed. And through Phytozome and the transcriptome data of this experiment, it can be seen that Gm20g008900 is only slightly expressed in flowers, and the expression level in other tissues is close to 0, so the remaining 5 homologous genes were analyzed by quantitative PCR. The results showed that GmLRX3 was highly expressed in soybean nodules, and the other 4 homologous genes (Gm05g232000, Gm08g039400, Gm10g245100, Gm20g149500) were expressed at very low levels in nodules ( Figure 4 ).

[0104] Based on the expression level and evolutionary relationship, stable transformed soybean plants lacking GmLRX3 and GmLRX4 were constructed using CRISPR / Cas9 technology. In the Gmlrx3 / 4 double mutant, GmLRX3 and GmLRX4 deleted 26 bp and 5 bp in the CDS region, respectively, both causing premature translation termination ( Figure 5 In A and B). To avoid the effects of the persistent presence of Cas9, Cas9 was isolated in the offspring of the Gmlrx3 / 4 double mutant. In addition, since Lotus japonicus and soybean are both legumes with NCRs deletions, the homologous gene of GmLRX4 in Lotus japonicus, LjLRX4, was also studied. Phenotypic observations were performed using a Lotus japonicus mutant with LORE1 inserted in the coding region of LjLRX4 ( Figure 5 Middle C).

[0105] Because GmLRX4 is localized in the perimycorrhizal cavity of the nodules, ultrathin sections were prepared from nodules of the Gmlrx3 / 4 double mutant and wild type in order to clearly observe the phenotype. Transmission electron microscopy analysis showed that the proportion of 5-10 bacteroids in the nodule symbionts of the Gmlrx3 / 4 double mutant was significantly increased (27% vs 5% of the wild type) ( Figure 6 Similarly, the symbionts of the Lotus japonicus Ljlrx4 mutant contained more bacteroids than the wild type ( Figure 6 These data suggest that the absence of GmLRX4 and GmLRX3 increases the number of bacteroids in the symbionts, which may be a conserved mechanism in legumes lacking NCRs.

[0106] To clarify the changes in bacteroids, transcriptome sequencing was performed on rhizobia grown in wild-type and mutant nodules ( Figure 7 ), compared with rhizobia in wild-type nodules, the expression of rhizobia cell division-related genes in the Gmlrx3 / 4 mutant was significantly upregulated, indicating that the increase in bacteroid number caused by the loss of GmLRX3 and GmLRX4 may be due to more active division of bacteroids.

[0107] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0108] The above-mentioned embodiments only express several implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application shall be subject to the attached claims, and the description and drawings may be used to interpret the contents of the claims.

Claims

1. Use of a recombinant expression vector comprising a nucleic acid molecule encoding a cell wall protein in regulating the number of rhizobia in a plant.

2. The use according to claim 1, characterized in that The recombinant expression vector regulates the number of rhizobia in the plant and thus regulates the nitrogen fixation efficiency of the plant's root nodules.

3. The use according to claim 1 or 2, characterized in that The cell wall protein includes at least one of GmLRX3 and GmLRX4.

4. A method for cultivating high-efficiency nodulation and nitrogen-fixing plants, characterized in that: The method comprises changing the content and / or activity of nucleic acid molecules encoding cell wall proteins in target plants, thereby effectively controlling the number of rhizobia and obtaining plants with high efficiency in nitrogen fixation; Wherein, the cell wall protein is optionally as defined in claim 3.

5. The method according to claim 4, characterized in that The method comprises increasing the expression level of a nucleic acid molecule encoding a cell wall protein in a target plant.

6. The method according to claim 5, characterized in that Increasing the expression level is achieved by at least one of the following methods: 1) increasing the copy number of the nucleic acid molecule; 2) expressing the nucleic acid molecule under the drive of a strong promoter; 3) increasing the regulatory element of the nucleic acid molecule to overexpress it, wherein the regulatory element is selected from the group consisting of an enhancer element, an element that improves mRNA stability, an element that enhances translation efficiency, and an element that enhances protein secretion; 4) Increase the ribosome binding sites of the nucleic acid molecule.

7. The method according to claim 6, characterized in that Increasing the expression level is achieved by introducing the nucleic acid molecule into the target plant.

8. The method according to claim 7, characterized in that The target plant is transfected with a recombinant expression vector containing a nucleic acid molecule encoding a cell wall protein.

9. The method according to claim 8, characterized in that The method satisfies one or more of the following conditions: i) introducing the nucleic acid molecule into the target plant by hairy root transformation; ii) Obtaining target plants with target gene knockout by stable transformation; iii) introducing the nucleic acid molecule into the underground part of the target plant; iv) introducing the nucleic acid molecule into the perimillary cavity of a target plant.

10. The method according to any one of claims 4 to 9, characterized in that: The plant is selected from the group consisting of Fabales, Cucurbitales, Rosales and Fagales.

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