Identification and application of a gene regulating the change of pH in symbiotic nodule

Abstract

The present application relates to identification and application of a gene regulating pH change in symbiotic nodule formation, and particularly provides Lotus japonicus HA1 gene or its encoded protein, and use of the HA1 gene or its encoded protein for improving agronomic traits of a plant, or for preparing a composition or preparation for improving agronomic traits of a plant, wherein the improved agronomic traits of the plant include (i) promoting nodule formation of rhizobia, (ii) improving nitrogen fixation ability, and / or (iii) improving quality and yield. It is first discovered in the present application that LjHA1 may participate in the process of symbiotic nodule formation by regulating pH change, and further has activities of (i) promoting nodule formation of rhizobia, (ii) improving nitrogen fixation ability, and / or (iii) improving quality and yield.

Description

Technical Field

[0001] This invention relates to the field of agronomy, specifically to the identification and application of a gene that regulates pH changes during symbiotic nodulation. Background Technology

[0002] Legumes are the third largest family of flowering plants, with members found worldwide. Most legumes form symbiotic nitrogen-fixing relationships with rhizobia in the soil, forming root nodules. These nodules contain rhizobia that convert atmospheric nitrogen into ammonia, which the plant uses for its growth. This allows legumes to grow in soils lacking mineral and organic nitrogen. This ability to naturally inject nitrogen into the soil makes legumes key species in natural and agro-ecosystems. In recent years, the continuous rise in fossil fuel prices has led to a sharp increase in the market price of chemical nitrogen fertilizers. Furthermore, the production and use of chemical nitrogen fertilizers are accompanied by greenhouse gas emissions. In addition, the utilization rate of industrial fertilizers in agricultural production is currently low, as 30%-50% of the applied nitrogen fertilizer cannot be utilized by plants and leaks into the environment, causing environmental problems such as eutrophication. Although symbiotic nitrogen fixation is the main source of nitrogen in agricultural systems, legumes (which fix approximately 50 million tons of nitrogen annually through symbiotic fixation) provide less than half the nitrogen from chemical fertilizers. Therefore, deepening the research on symbiotic nitrogen fixation in legumes is of great significance for agricultural production and environmental protection.

[0003] The establishment of a symbiotic relationship between legumes and rhizobia involves rhizobium infection and nodule formation, and the formation of mature nitrogen-fixing nodules requires the coordinated action of these two processes in time and space. In all legumes studied, approximately 75% chose root hair infection as the mode of transmission. Upon responding to nodulation factor signals released by rhizobia, the root hairs of legumes curl and deform, enveloping the rhizobia. Once inside the root hair, the enveloped rhizobia extend downwards along pre-formed tubular infection lines within the root hair, eventually entering the nodule organ in the root cortex and being released. Currently, research on the root hair infection process is incomplete, and it is unclear how environmental factors influence this process.

[0004] Soil acidification is a major form of global soil degradation, characterized by a continuous decrease in soil pH and an increase in exchangeable acidity. Strengthening research on soil acidification regulation and acidic soil improvement is of great significance for improving soil quality and productivity. + -ATPases regulate pH changes inside and outside the plant cell. Plant H + -ATPases, including phosphorylated intermediates (P-type) and vacuolar (V-type) H-types located in the plasma membrane. +-ATPases are important ATP-driven proton pumps that generate membrane potential and provide the proton driving potential for secondary active transport. Studies have shown that H + -ATPases play multiple roles in signal transduction during cell expansion, regulation of intracellular pH, plant response to salt stress, and regulation of stomatal opening.

[0005] However, no information about H has been found yet. + - Reports on ATPase regulation of symbiotic nodulation. [Regarding] plant H... + Research on ATPases plays an important role in improving the ability of crops to adapt to acidified soils.

[0006] Therefore, there is an urgent need in this field to develop new methods to improve plant symbiotic nodulation. Summary of the Invention

[0007] The purpose of this invention is to provide a new method for improving plant symbiotic nodulation.

[0008] A first aspect of the present invention provides an isolated polypeptide selected from the group consisting of:

[0009] (a) A polypeptide with the amino acid sequence shown in SEQ ID No. 2;

[0010] (b) A derivative polypeptide formed by adding a tag sequence, signal sequence or secretion signal sequence to SEQ ID NO.2, and having (i) the ability to promote rhizobium nodulation; (ii) the ability to improve nitrogen fixation; and / or (iii) the activity of improving quality and yield;

[0011] (c) A derivative polypeptide having an amino acid sequence that is ≥95% homology to the amino acid sequence shown in SEQ ID NO.2, preferably ≥98%, more preferably ≥99%, and having (i) the ability to promote rhizobium nodulation; (ii) the ability to improve nitrogen fixation; and / or (iii) the activity of improving quality and yield.

[0012] In another preferred embodiment, (c) further includes a derivative polypeptide formed by substituting, deleting or adding one or more (preferably 1-10, more preferably 1-5) amino acid residues of the polypeptide of the amino acid sequence shown in SEQ ID NO.2, and having (i) the ability to promote rhizobium nodulation; (ii) the ability to improve nitrogen fixation; and / or (iii) the activity of improving quality and yield.

[0013] A second aspect of the present invention provides a polynucleotide comprising a nucleotide sequence as shown in SEQ ID NO:1.

[0014] In another preferred embodiment, the polynucleotide is selected from the group consisting of:

[0015] (a) A polynucleotide encoding the polypeptide shown in SEQ ID NO.:2;

[0016] (b) Polynucleotides with sequences as shown in SEQ ID NO.:1;

[0017] (c) A polynucleotide whose nucleotide sequence is ≥80% homology (preferably ≥90%) to the sequence shown in SEQ ID NO.:1 and encodes the polypeptide shown in SEQ ID NO.:2;

[0018] (d) and any of the polynucleotides complementary to those described in (a)-(c).

[0019] In another preferred embodiment, the polynucleotide is selected from the group consisting of DNA sequences, RNA sequences, or combinations thereof.

[0020] In another preferred embodiment, the polynucleotide further comprises, flanking the ORF of the nucleotide sequence, an auxiliary element selected from the group consisting of: signal peptides, secretory peptides, tag sequences (such as 6His), or combinations thereof.

[0021] In another preferred embodiment, the polynucleotide further comprises a promoter operatively linked to the ORF sequence of the nucleotide sequence.

[0022] In another preferred embodiment, the promoter is selected from the group consisting of: constitutive promoters, tissue-specific promoters, inducible promoters, or strong promoters.

[0023] A third aspect of the present invention provides a carrier containing the polynucleotide described in the second aspect of the present invention.

[0024] In another preferred embodiment, the carrier includes an expression carrier, a shuttle carrier, and an integration carrier.

[0025] A fourth aspect of the present invention provides a host cell containing the vector described in the third aspect of the present invention, or having the polynucleotides described in the second aspect of the present invention integrated into its genome.

[0026] In another preferred embodiment, the host cell is a eukaryotic cell, such as a yeast cell or a plant cell.

[0027] In another preferred embodiment, the plant is a symbiotic nodular plant.

[0028] In another preferred embodiment, the plant is a plant that forms nodules in symbiosis with rhizobia.

[0029] In another preferred embodiment, the plant is a legume, including soybean, alfalfa, and bird's eye.

[0030] In another preferred embodiment, the host cell is a prokaryotic cell, such as Escherichia coli.

[0031] The fifth aspect of the present invention provides the use of the polypeptide described in the first aspect of the present invention, the polynucleotide described in the second aspect of the present invention, and the carrier described in the third aspect of the present invention for improving the agronomic traits of plants, or for preparing a composition or formulation for improving the agronomic traits of plants, said composition or formulation for improving the agronomic traits of plants including (i) promoting the ability of rhizobia to form nodules; (ii) improving nitrogen fixation capacity; and / or (iii) improving quality and yield.

[0032] In another preferred embodiment, the plant is a symbiotic nodular plant.

[0033] In another preferred embodiment, the plant is a plant that forms nodules in symbiosis with rhizobia.

[0034] In another preferred embodiment, the plant is a legume, including soybean, alfalfa, and bird's eye.

[0035] In another preferred embodiment, the improved plant agronomic traits also include increased stress resistance, quality, yield, and protein content.

[0036] The sixth aspect of the present invention provides the use of the HA1 gene or the protein encoded therein for improving agronomic traits of plants, or for preparing a composition or formulation for improving agronomic traits of plants, said improved agronomic traits including (i) promoting the ability of rhizobia to form nodules; (ii) improving nitrogen fixation capacity; and / or (iii) improving quality and yield.

[0037] In another preferred embodiment, the improved plant agronomic traits also include increased stress resistance, quality, yield, and protein content.

[0038] In another preferred embodiment, the HA1 gene or its encoded protein is induced by rhizobia, thereby promoting the interaction between the plant and rhizobia, promoting rhizobia infection, and promoting the ability of the plant and rhizobia to form nitrogen-fixing root nodules in symbiosis.

[0039] In another preferred embodiment, the rhizobium is a slow-growing type of Rhizobium loulatum.

[0040] In another preferred embodiment, the rhizobium is the slow-growing type of Mesorhizobium loti R7A / lacZ.

[0041] In another preferred embodiment, the HA1 gene or its encoded protein is derived from legumes or non-legumes (including grasses and cruciferous plants).

[0042] In another preferred embodiment, the HA1 gene or its encoded protein is derived from one or more plants selected from the group consisting of: birdsfoot root, alfalfa, soybean, rice, maize, and Arabidopsis thaliana, with birdsfoot root being preferred.

[0043] In another preferred embodiment, the nucleotide sequence of the HA1 gene is selected from the group consisting of:

[0044] (a) A polynucleotide encoding a polypeptide as shown in any of SEQ ID NO.:2, 5, 6, or 8;

[0045] (b) Polynucleotides with sequences as shown in any of SEQ ID NO.: 1, 3, 4, 7;

[0046] (c) A polynucleotide whose nucleotide sequence is ≥80% homology (preferably ≥90%) to any of the sequences shown in SEQ ID NO.:1, 3, 4, 7, and encodes a polypeptide shown in any of SEQ ID NO.:2, 5, 6, 8;

[0047] (d) and any of the polynucleotides complementary to those described in (a)-(c).

[0048] In another preferred embodiment, the HA1 gene or its encoded protein is derived from the root of *Lotus styrax* or a variant thereof.

[0049] In another preferred embodiment, the HA1 gene includes a wild-type HA1 gene and a mutant HA1 gene.

[0050] In another preferred embodiment, the mutants include mutants in which the function of the encoded protein remains unchanged (i.e., the function is the same as or substantially the same as the wild-type encoded protein) and mutants in which the function is enhanced.

[0051] In another preferred embodiment, the polypeptide encoded by the mutant HA1 gene is the same as or substantially the same as the polypeptide encoded by the wild-type HA1 gene.

[0052] In another preferred embodiment, the mutant HA1 gene comprises polynucleotides with ≥80% (preferably ≥90%, more preferably ≥95%, even more preferably ≥98% or 99%) homology to the wild-type HA1 gene.

[0053] In another preferred embodiment, the mutant HA1 gene comprises a polynucleotide with 1-60 (preferably 1-30, more preferably 1-10) nucleotides truncated or added to the 5' and / or 3' ends of the wild-type HA1 gene.

[0054] In another preferred embodiment, the amino acid sequence of the HA1 protein is selected from the group consisting of:

[0055] (i) A polypeptide having any of the amino acid sequences shown in SEQ ID NO.:2, 5, 6, 8;

[0056] (ii) A polypeptide derived from (i) that has the function of improving the agronomic traits of plants, formed by substituting, deleting or adding one or more (e.g., 1-10) amino acid residues of an amino acid sequence as shown in any of SEQ ID NO.:2, 5, 6, 8; or

[0057] (iii) A polypeptide having the agronomic trait function of the plant being improved, having an amino acid sequence that is ≥80% homology (preferably ≥90%, more preferably ≥95% or ≥98%) with any of the amino acid sequences shown in SEQ ID NO.:2, 5, 6, 8.

[0058] In another preferred embodiment, the composition is an agricultural composition.

[0059] In another preferred embodiment, the composition comprises (a) the HA1 gene or its encoded protein; and (b) an agronomically acceptable vector.

[0060] In another preferred embodiment, the dosage form of the composition or preparation is selected from the group consisting of solutions, emulsions, suspensions, powders, foams, pastes, granules, aerosols, or combinations thereof.

[0061] In another preferred embodiment, the plant is a nodulation plant that forms a symbiotic relationship with rhizobia.

[0062] In another preferred embodiment, the plant is a legume, including soybean, alfalfa, and bird's eye.

[0063] A seventh aspect of the present invention provides a composition comprising:

[0064] (a) The HA1 gene or its encoded protein;

[0065] (b) An agriculturally acceptable carrier.

[0066] In another preferred embodiment, the HA1 gene or its encoded protein is derived from legumes, grasses, or cruciferous plants.

[0067] In another preferred embodiment, the HA1 gene or its encoded protein is derived from one or more plants selected from the group consisting of soybean, alfalfa, birdsfoot, maize, rice, wheat, and Arabidopsis thaliana, with birdsfoot being the preferred source.

[0068] In another preferred embodiment, the nucleotide sequence of the HA1 gene is selected from the group consisting of:

[0069] (a) A polynucleotide encoding a polypeptide as shown in any of SEQ ID NO.:2, 5, 6, or 8;

[0070] (b) Polynucleotides with sequences as shown in any of SEQ ID NO.: 1, 3, 4, 7;

[0071] (c) A polynucleotide whose nucleotide sequence is ≥80% homology (preferably ≥90%) to any of the sequences shown in SEQ ID NO.:1, 3, 4, 7, and encodes a polypeptide shown in any of SEQ ID NO.:2, 5, 6, 8;

[0072] (d) and any of the polynucleotides complementary to those described in (a)-(c).

[0073] In another preferred embodiment, the HA1 gene or its encoded protein is derived from the root of *Lotus styrax* or a variant thereof.

[0074] In another preferred embodiment, the composition comprises an agricultural composition.

[0075] In another preferred embodiment, the dosage form of the composition is selected from the group consisting of solutions, emulsions, suspensions, powders, foams, pastes, granules, aerosols, or combinations thereof.

[0076] In another preferred embodiment, the composition contains 0.0001-10 wt%, preferably 0.1-1 wt%, of component (a) based on the total weight of the composition.

[0077] In another preferred embodiment, the composition further includes other substances for (i) promoting the nodulation ability of rhizobia; (ii) improving nitrogen fixation ability; and / or (iii) improving quality and yield.

[0078] In another preferred embodiment, the other substances used to (i) promote the nodulation ability of rhizobia; (ii) improve nitrogen fixation ability; and / or (iii) improve quality and yield include: flavonoids such as daidzein, genistein, or phenolic acids such as phenoxyacetic acid (POA).

[0079] The eighth aspect of the present invention provides the use of the composition described in the seventh aspect of the present invention for (i) promoting the nodulation ability of plant rhizobia; (ii) improving the nitrogen fixation ability of plants; and / or (iii) improving plant quality and yield.

[0080] In another preferred embodiment, the composition is also used to improve stress resistance quality yield and protein content.

[0081] A ninth aspect of the present invention provides a method for improving the agronomic traits of plants, the method comprising the step of introducing the HA1 gene into plant cells, plant seeds, plant tissues, plant parts or plants; the improved agronomic traits of plants include (i) promoting the ability of rhizobia to form nodules; (ii) improving nitrogen fixation capacity; and / or (iii) improving quality and yield.

[0082] In another preferred embodiment, the improved plant agronomic traits also include increased stress resistance, quality, yield, and protein content.

[0083] In another preferred embodiment, the method further includes the step of co-culturing the plant with rhizobia.

[0084] In another preferred embodiment, the HA1 gene is derived from legumes, grasses, or cruciferous plants.

[0085] In another preferred embodiment, the HA1 gene is derived from one or more plants selected from the group consisting of soybean, alfalfa, birdsfoot root, maize, rice, wheat, and Arabidopsis thaliana, with birdsfoot root being preferred.

[0086] In another preferred embodiment, the introduction of the HA1 gene according to the present invention includes the step of expressing the HA1 gene in plant cells, plant seeds, plant tissues, plant parts or plants, for example, by expressing the HA1 gene through an expression vector, or by integrating the HA1 gene into the plant genome for expression.

[0087] In another preferred embodiment, the method includes the steps of:

[0088] (i) Provide a plant cell, plant seed, plant tissue, plant part, or plant; and

[0089] (ii) The HA1 gene sequence is introduced into the plant cell, plant seed, plant tissue, plant part or plant to obtain a transgenic plant cell, plant seed, plant tissue, plant part or plant.

[0090] In another preferred embodiment, the method includes the steps of:

[0091] (a) Agrobacterium providing an expression vector carrying the HA1 gene sequence;

[0092] (b) Contacting plant cells, tissues or organs with Agrobacterium in step (a) to transfer the gene sequence of HA1 into the plant cells, tissues or organs;

[0093] (c) Select plant cells, tissues, or organs that have been transfected with the HA1 gene sequence; and

[0094] (d) Regenerate the plant cells, tissues or organs from step (c) into a plant.

[0095] In another preferred embodiment, the method includes the steps of:

[0096] (a) Introducing a foreign construct into plant cells, wherein the construct contains a foreign HA1 gene sequence, thereby obtaining plant cells in which the foreign construct has been introduced;

[0097] (b) Regenerate plants from the plant cells containing the introduced exogenous construct obtained in the previous step; and (c) optionally identify the regenerated plants to obtain plants that have (i) the ability to promote rhizobium nodulation; (ii) the ability to improve nitrogen fixation; and / or (iii) the activity of improving quality and yield.

[0098] In another preferred embodiment, the exogenous HA1 gene sequence further includes a promoter and / or terminator operatively linked to the ORF sequence.

[0099] In another preferred embodiment, the promoter is selected from the group consisting of constitutive promoters, tissue-specific promoters, inducible promoters, and strong promoters, with strong promoters being preferred.

[0100] In another preferred embodiment, the plant includes legumes.

[0101] In another preferred embodiment, the plant includes soybean, alfalfa, and bird's eye root.

[0102] The tenth aspect of this invention provides a method for preparing genetically engineered plant tissues or plant cells, comprising the steps of:

[0103] The HA1 gene or its encoded protein is introduced into plant cells or plant tissues to obtain genetically engineered plant tissues or plant cells.

[0104] In another preferred embodiment, the genetic engineering includes transgenic engineering.

[0105] In another preferred embodiment, the method further includes introducing the HA1 gene or its encoded protein into plant tissues or plant cells.

[0106] The eleventh aspect of this invention provides a method for preparing plants with improved traits, comprising the steps of:

[0107] The genetically engineered plant tissues or plant cells prepared by the method described in the tenth aspect of the present invention are regenerated into plant bodies, thereby obtaining plants with improved traits.

[0108] In another preferred embodiment, the traits include (i) promoting the ability of rhizobia to form nodules; (ii) improving nitrogen fixation capacity; and / or (iii) improving quality and yield.

[0109] In another preferred embodiment, the traits also include improved stress resistance, quality yield, and protein content.

[0110] In another preferred embodiment, the method further includes the step of co-culturing the plant with rhizobia.

[0111] The twelfth aspect of the present invention provides a genetically engineered plant in which the HA1 gene or its encoded protein is introduced, or the plant is prepared using the method described in the eleventh aspect of the present invention.

[0112] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0113] The following figures are used to illustrate specific embodiments of the present invention and are not intended to limit the scope of the invention as defined by the claims.

[0114] Figure 1 The H in Arabidopsis thaliana, Alfalfa, and Lobelia chinensis was shown. + - ATPase AHA protein phylogenetic tree. The phylogenetic tree was derived using the maximum likelihood method based on the JTT matrix model [1]. The initial tree for the heuristic search was obtained automatically by applying adjacency connections and the BioNJ algorithm to the pairwise distance matrix estimated using the JTT model, and then selecting the topology with the better log-likelihood value. The tree was drawn to scale, and the branch length was measured by the number of substitutions at each site. The analysis involved 37 amino acid sequences. The evolutionary analysis was performed in MEGA7 [2].

[0115] Figure 2 The expression pattern of HA1 in *Lotus sibiricum* roots is shown. (A) qRT-PCR analysis of LjHA1 expression in wild-type roots at 1, 4, and 7 days after inoculation with *M. loli* R7A / lacZ. An asterisk indicates a significant difference between the control and rhizobium treatments at different time points. (B) pHA1:GUS expression pattern in roots and nodules. Transformed roots were stained with X-Gluc, and blue indicates the location of GUS expression. Bar = 100 μm.

[0116] Figure 3 The symbiotic nodulation phenotype of the HA1 mutant in *Lobelia chinensis* is shown. (AB) Number of root nodules in LjHA1 mutant roots after inoculation (5 days). Bar = 100 μm. (CD) Number of root nodules in LjHA1 mutant roots after inoculation (2 weeks). Asterisks indicate significant differences, *P<0.05, **P<0.01, t-test. Bar = 1 mm.

[0117] Figure 4 This study shows the symbiotic infection phenotype of the HA1 mutant in *Lobelia chinensis* under different pH conditions. The infection phenotype of plants was statistically analyzed 5 days after inoculation with *M. loti* rhizobia. An asterisk indicates a significant difference; *P<0.05, **P<0.01, t-test. Detailed Implementation

[0118] Through extensive and in-depth research, the inventors, through the study and screening of numerous plant trait loci, have for the first time discovered in *Lotus japonicus* that LjHA1 is specifically induced by rhizobia; mutations in LjHA1 lead to a reduction in infection events and nodule number. Furthermore, it was found that compared to normal pH environments, LjHA1 infection events are further reduced in acidic or alkaline environments. These results suggest that LjHA1 may participate in the symbiotic nodulation process by regulating pH changes, and further (i) promote the nodulation ability of rhizobia; (ii) enhance nitrogen fixation capacity; and / or (iii) improve quality and yield. Based on these findings, the inventors completed this invention.

[0119] HA1 gene

[0120] As used in this article, the term HA1 gene is H + -ATPase. As used herein, the terms "HA1 gene of the present invention" and "HA1 gene" are used interchangeably and both refer to the HA1 gene or a variant thereof derived from plants (such as legumes, preferably Gallus gallus domesticus).

[0121] In a preferred embodiment, the nucleotide sequence of the HA1 gene of the present invention is shown in SEQ ID NO.: 1, 3, and 4. Variants of the HA1 gene of the present invention can be obtained by inserting or deleting regulatory regions, performing random or site-directed mutations, etc.

[0122] The present invention also includes nucleic acids having 50% or more (preferably 60%, 70%, 80%, more preferably 90%, more preferably 95%, most preferably 98%, such as 99%) homology to the preferred gene sequences of the present invention (SEQ ID NO.: 1, 3, 4, 7), said nucleic acids also effectively regulating the traits of plants such as soybean, alfalfa, and birdsfoot root. "Homology" refers to the level of similarity (i.e., sequence similarity or identity) between two or more nucleic acids according to the percentage of identical positions.

[0123] In this invention, the nucleotide sequences in SEQ ID NO.:1, 3, 4, and 7 can be modified by substitution, deletion, or addition of one or more (usually 1-90, preferably 1-60, more preferably 1-20, and most preferably 1-10), and by adding several (usually up to 60, preferably up to 30, more preferably up to 10, and most preferably up to 5) nucleotides to the 5' and / or 3' ends to generate derived sequences of SEQ ID NO.:1, 3, 4, and 7. Due to the degeneracy of the codons, even with low homology to SEQ ID NO.:1, 3, 4, and 7, the amino acid sequences shown in SEQ ID NO.:2, 5, 6, and 8 can be encoded.

[0124] Furthermore, the meaning of "the nucleotide sequences in SEQ ID NO.:1, 3, 4, 7 having undergone substitution, deletion, or addition of at least one nucleotide-derived sequence" also includes nucleotide sequences capable of hybridizing with the nucleotide sequences shown in SEQ ID NO.:1, 3, 4, 7 under moderately stringent conditions, and preferably under highly stringent conditions. These variations include (but are not limited to): deletions, insertions, and / or substitutions of several nucleotides (typically 1-90, preferably 1-60, more preferably 1-20, and most preferably 1-10), and additions of several nucleotides (typically up to 60, preferably up to 30, more preferably up to 10, and most preferably up to 5) at the 5' and / or 3' ends.

[0125] It should be understood that although the genes provided in the examples of this invention are derived from *Lotus sibiricum*, HA1 gene sequences derived from other similar plants (especially those belonging to the same legume family as *Lotus sibiricum* or other families or genera with high homology to *Lotus sibiricum*) that have a certain degree of homology (conservatism, such as having more than 80%, such as 85%, 90%, 95%, or even 98% sequence identity) with the sequences of this invention (preferably, sequences such as SEQ ID NO.: 1, 3, 4, 7) are also included within the scope of this invention. As long as those skilled in the art can easily isolate the sequence from other plants based on the information provided in this application after reading this application, the methods and tools for comparing sequence identity are also well known in the art, such as BLAST.

[0126] The polynucleotides of this invention can be in DNA or RNA form. The DNA form includes DNA, genomic DNA, or artificially synthesized DNA, which can be single-stranded or double-stranded. The DNA can be a coding strand or a non-coding strand. The coding region sequence encoding the mature polypeptide can be identical to or a degenerate variant of the coding region sequences shown in SEQ ID NO.:1, 3, 4, and 7.

[0127] Polynucleotides encoding mature polypeptides include: coding sequences that encode only the mature polypeptide; coding sequences of the mature polypeptide and various additional coding sequences; coding sequences of the mature polypeptide (and optional additional coding sequences) and non-coding sequences.

[0128] The term "polynucleotide encoding a polypeptide" can refer to a polynucleotide that encodes the polypeptide, or it can also include polynucleotides that include additional coding and / or non-coding sequences. This invention also relates to variants of the aforementioned polynucleotides that encode fragments, analogs, and derivatives of polyglycosides or polypeptides having the same amino acid sequence as those of this invention. These polynucleotide variants can be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants, and insertion variants. As is known in the art, an allelic variant is a substitution of a polynucleotide, which may be a substitution, deletion, or insertion of one or more nucleotides, but does not substantially alter the function of the polypeptide it encodes.

[0129] The present invention also relates to polynucleotides that hybridize with the above-described sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that hybridize with the polynucleotides described herein under stringent conditions. In the present invention, “stringent conditions” means: (1) hybridization and elution at lower ionic strength and higher temperatures, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) hybridization with a denaturing agent, such as 50% (v / v) formamide, 0.1% fetal bovine serum / 0.1% Ficoll, 42°C, etc.; or (3) hybridization only occurs when the identity between the two sequences is at least 90%, preferably at least 95%.

[0130] The full-length nucleotide sequence or fragments of the HA1 gene of this invention can generally be obtained by PCR amplification, recombination, or artificial synthesis. For PCR amplification, primers can be designed based on the nucleotide sequences disclosed in this invention, especially the open reading frame sequences, and the relevant sequences can be amplified using commercially available DNA libraries or cDNA libraries prepared according to conventional methods known to those skilled in the art. When the sequence is long, two or more PCR amplifications are often required, and then the fragments amplified from each amplification are spliced ​​together in the correct order. Once the relevant sequence is obtained, it can be obtained in large quantities using recombination. Typically, it is cloned into a vector, transformed into cells, and then the relevant sequence is isolated from the proliferated host cells using conventional methods.

[0131] Furthermore, the relevant sequences can be synthesized artificially, especially when the fragment length is short. Typically, long fragments are obtained by first synthesizing multiple small fragments and then ligating them. Currently, the DNA sequence encoding the protein of the present invention (or a fragment thereof, or a derivative thereof) can be obtained entirely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. Furthermore, mutations can be introduced into the protein sequence of the present invention through chemical synthesis.

[0132] In a preferred embodiment, the nucleotide sequence of the HA1 gene (LjHA1) is as follows:

[0133] LjHA1 CDS (2907bp):

[0134]

[0135] In a preferred embodiment, the nucleotide sequence of the HA1 gene (MtHA1) is as follows:

[0136] MtHA1 CDs (2901bp):

[0137]

[0138] Nucleotide sequence of the HA1 gene in soybean:

[0139]

[0140] The nucleotide sequence of LjHA1 corresponding to the Arabidopsis thaliana homolog AHA:

[0141]

[0142] HA1 gene-encoded polypeptide

[0143] As used herein, the terms "polypeptide of the present invention" and "protein encoded by the HA1 gene" are used interchangeably and refer to a polypeptide of HA1 derived from plants (such as soybean, alfalfa, birdsfoot root, corn, rice, wheat, Arabidopsis thaliana, preferably birdsfoot root), or a polypeptide of HA1 and its variants. In a preferred embodiment, a typical amino acid sequence of the polypeptide of the present invention is shown in SEQ ID NO. 2, 5, 6, 8.

[0144] This invention relates to an HA1 polypeptide and its variants for improving plant agronomic traits. In a preferred embodiment of the invention, the amino acid sequence of the polypeptide is shown in SEQ ID NO. 2, 5, 6, 8. The polypeptide of the present invention can effectively improve the traits of plants (such as birdsfoot root, soybean, and alfalfa), for example (i) promoting the nodulation ability of rhizobia; (ii) improving nitrogen fixation ability; and / or (iii) improving quality and yield.

[0145] The present invention also includes polypeptides or proteins having the same or similar functions and having 50% or more (preferably 60% or more, 70% or more, 80% or more, more preferably 90% or more, more preferably 95% or more, most preferably 98% or more, such as 99%) homology to the sequences shown in SEQ ID NO.:2, 5, 6, 8 of the present invention.

[0146] The “same or similar functions” mainly refer to: “regulating the traits of plants or crops (such as soybeans, alfalfa, and birdsfoot root), such as (i) promoting the nodulation ability of rhizobia; (ii) improving nitrogen fixation ability; and / or (iii) improving quality and yield.”

[0147] The polypeptides of the present invention can be recombinant polypeptides, natural polypeptides, or synthetic polypeptides. The polypeptides of the present invention can be naturally purified products, chemically synthesized products, or produced from prokaryotic or eukaryotic hosts (e.g., bacteria, yeast, higher plants, insects, and mammalian cells) using recombinant technology. Depending on the host used in the recombinant production protocol, the polypeptides of the present invention can be glycosylated or non-glycosylated. The polypeptides of the present invention may or may not include an initial methionine residue.

[0148] The present invention also includes plant-derived HA1 protein fragments and analogs having the protein activity of the present invention. As used herein, the terms “fragment” and “analyte” refer to polypeptides that substantially retain the same biological function or activity as the natural HA1 protein of the present invention.

[0149] The polypeptide fragments, derivatives, or analogs of the present invention may be: (i) polypeptides in which one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code; or (ii) polypeptides having substituent groups in one or more amino acid residues; or (iii) polypeptides formed by fusing a mature polypeptide with another compound (e.g., a compound that extends the half-life of the polypeptide, such as polyethylene glycol); or (iv) polypeptides formed by fusing an additional amino acid sequence to this polypeptide sequence (e.g., a leader sequence or secretion sequence or a sequence used to purify this polypeptide or a proteogen sequence, or a fusion protein). These fragments, derivatives, and analogs, as defined herein, are within the scope well known to those skilled in the art.

[0150] In this invention, the polypeptide variants are derived sequences obtained by substituting, deleting, or adding at least one amino acid, as shown in SEQ ID NO. 2, 5, 6, and 8, with a number of substitutions (typically 1-60, preferably 1-30, more preferably 1-20, and most preferably 1-10), deletions, or additions, and by adding one or more amino acids (typically up to 20, preferably up to 10, and more preferably up to 5) to the C-terminus and / or N-terminus. For example, in the protein, substitution with amino acids of similar or comparable properties generally does not alter the protein's function, and adding one or more amino acids to the C-terminus and / or N-terminus generally does not alter the protein's function either. These conserved variations are preferably generated by substitutions according to Table I.

[0151] Table I

[0152] The initial residues Representative substitution Preferred replacement Ala(A) Val; Leu; Ile Val Arg(R) Lys;Gln;Asn Lys Asn(N) Gln; His; Lys; Arg Gln Asp(D) Glu Glu Cys(C) Ser Ser Gln(Q) Asn Asn Glu(E) Asp Asp Gly(G) Pro; Ala Ala His(H) Asn; Gln; Lys; Arg Arg Ile(I) Leu; Val; Met; Ala; Phe Leu Leu(L) Ile; Val; Met; Ala; Phe Ile Lys(K) Arg;Gln;Asn Arg Met(M) Leu; Phe; Ile Leu Phe(F) Leu; Val; Ile; Ala; Tyr Leu Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Ser Ser Trp(W) Tyr; Phe Tyr Tyr(Y) Trp; Phe; Thr; Ser Phe Val(V) Ile; Leu; Met; Phe; Ala Leu

[0153] This invention also includes analogs of the claimed proteins. These analogs may differ from the natural SEQ ID NO.:2, 5, 6, 8 in either amino acid sequence differences, or in the form of modifications that do not affect the sequence, or both. These protein analogs include natural or induced genetic variants. Induced variants can be obtained by various techniques, such as random mutagenesis through radiation or exposure to a mutagen, or by site-directed mutagenesis or other known biochemical techniques. Analogs also include those having residues different from natural L-amino acids (e.g., D-amino acids), and those having non-naturally occurring or synthetic amino acids (e.g., β, γ-amino acids). It should be understood that the proteins of this invention are not limited to the representative proteins exemplified above.

[0154] Modifications (which typically do not alter the primary structure) include chemically derived forms of proteins, such as acetate or carboxylation, either in vivo or in vitro. Modifications also include glycosylation, such as those occurring during protein synthesis and processing. This modification can be accomplished by exposing the protein to glycosylating enzymes, such as mammalian glycosylation or deglycosylation enzymes. Modifications also include sequences containing phosphorylated amino acid residues, such as phosphotyrosine, phosphotyserine, and phosphotythreonine.

[0155] In a preferred embodiment, the amino acid sequence of HA1 (LjHA1) is as follows:

[0156] LjHA1 protein (968aa):

[0157] *(SEQ ID NO.2)

[0158] In a preferred embodiment, the amino acid sequence of HA1 (MtHA1) is as follows:

[0159] MtHA1 protein (967aa):

[0160] *(SEQ ID NO.5)

[0161] The HA1 amino acid sequence of soybean:

[0162] *(SEQ ID NO.6)

[0163] Amino acid sequence of the homologous gene AHA of Arabidopsis thaliana corresponding to LjHA1:

[0164] MTDIEALKAITTESIDLENVPVEEVFQHLKCTKEGLTSNEVQERLTLFGYNKLEEKKESKILKFLGF

[0165] MWNPLSWVMEAAALMAIGLAHGGGKPADYHDFVGIVVLLLINSTISFVEENNAGNAAAALMAQLAPK

[0166] AKAVRDGKWNEIDAAELVPGDIVSIKLGDIIPADARLLEGDPLKIDQATLTGESLPVTKNPGASVYS

[0167] GSTCKQGEIEAVVIATGVHTFFGKAAHLVDSTTHVGHFQKVLTAIGNFCICSIAVGMAIEIVVIYGL

[0168] QKRGYRVGIDNLLVLLIGGIPIAMPTVLSVTMAIGAHRLAQQGAITKRMTAIEEMAGMDVLCSDKTG

[0169] TLTLNKLSVDKNLIEVFKRGIDRDMAVLMAARAARLENQDAIDTAIVSMLSDPKEARAGIKELHFLP

[0170] FSPANRRTALTYLDGEGKMHRVSKGAPEEILDMAHNKLEIKEKVHATIDKFAERGLRSLGLAYQEVP

[0171] DGDVKGEGGPWDFVALLPLFDPPRHDSAQTIERALHLGVSVKMITGDQLAIAKETGRRLGMGTNMYP

[0172] SSSLLSDNNTEGVSVDELIENADGFAGVFPEHKYEIVKRLQSRKHICGMTGDGVNDAPALKKADIGI

[0173] AVDDATDAARGASDIVLTEPGLSVIISAVLTSRAIFQRMKNYTIYAVSITIRIVMGFMLLCVFWEFD

[0174] FPPFMVLVIAILNDGTIMTISKDRVKPSPTPDCWKLKEIFATGVVLGAYLAIMTVVFFWAAYETNFF

[0175] HNIFHVRNFNQHHFKMKDKKVAAHLNEQMASAVYLQVSTISQALIFVTRSRSWSFVERPGFLLVIAF

[0176] LIAQLVASVISAMANWPFAGIRSIGWGWTGVIWIFNIVTYMLLDPIKFLVRYALSGKSWDRMVEGRT

[0177] ALTGKKNFGQEERMAAWATEKRTQHGLETGQKPVYERNSATELNNMAEEAKRRAEIARMRELQTLKGKVESAAKLKGYDLEDPNSNNYTI*(SEQ ID NO.8)

[0178] expression carrier

[0179] The present invention also relates to vectors containing the polynucleotides of the present invention, host cells generated by genetic engineering using the vectors of the present invention or the coding sequences of mutant proteins of the present invention, and methods for generating the polypeptides of the present invention via recombinant technology.

[0180] Using conventional recombinant DNA technology, the polynucleotide sequence of this invention can be used to express or produce the protein or its variants described herein. Generally, the following steps are involved:

[0181] (1) Transform or transduce suitable host cells with a polynucleotide encoding the protein of the present invention or a variant thereof, or with a recombinant expression vector containing the polynucleotide;

[0182] (2) Host cells cultured in a suitable culture medium;

[0183] (3) Isolate and purify proteins from culture media or cells.

[0184] This invention also provides a recombinant vector comprising the gene of this invention. As a preferred embodiment, the recombinant vector contains a multiple cloning site or at least one restriction enzyme site downstream of the promoter. When it is necessary to express the target gene of this invention, the target gene is ligated into a suitable multiple cloning site or restriction enzyme site, thereby operatively linking the target gene to the promoter. As another preferred embodiment, the recombinant vector comprises (from 5' to 3' direction): a promoter, a target gene, and a terminator. If desired, the recombinant vector may further comprise elements selected from the group consisting of: a 3' polynucleotide signal; a non-translated nucleic acid sequence; a transport and targeting nucleic acid sequence; an resistance selection marker (dihydrofolate reductase, neomycin resistance, hygromycin resistance, and fluorescent proteins, etc.); an enhancer; or an operator.

[0185] In this invention, a polynucleotide sequence encoding a protein can be inserted into a recombinant expression vector. The term "recombinant expression vector" refers to bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors well-known in the art. Any plasmid and vector can be used as long as it can replicate and remain stable within the host. An important characteristic of expression vectors is that they typically contain an origin of replication, a promoter, a marker gene, and translational control elements.

[0186] Methods well known to those skilled in the art can be used to construct expression vectors containing the protein-coding DNA sequence of the present invention and suitable transcription / translation control signals. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, etc. When constructing recombinant expression vectors using the genes of the present invention, any type of enhancing, constitutive, tissue-specific, or inducible promoter can be added before its transcription initiation nucleotide.

[0187] The DNA sequence can be efficiently ligated to an appropriate promoter in the expression vector to direct mRNA synthesis. Representative examples of these promoters include: the *E. coli* lac or trp promoter; the *λ* phage PL promoter; eukaryotic promoters including the CMV immediate early promoter, the HSV thymidine kinase promoter, early and late SV40 promoters, retroviral LTRs, and other known promoters that control gene expression in prokaryotic or eukaryotic cells or their viruses. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

[0188] Vectors including the genes, expression cassettes, or other components of this invention can be used to transform suitable host cells to enable the host to express proteins. Host cells can be prokaryotic cells, such as *Escherichia coli*, *Streptomyces*, or *Agrobacterium*; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as plant cells. Those skilled in the art will understand how to select appropriate vectors and host cells. Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryote (such as *E. coli*), competent cells capable of uptake DNA can be harvested after the exponential growth phase and treated with CaCl2, the steps of which are well known in the art. Another method is to use MgCl2. If desired, transformation can also be performed using electroporation. When the host is a eukaryote, DNA transfection methods such as calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, and liposome packaging can be used.

[0189] Transformed plants can also be achieved using methods such as Agrobacterium-mediated transformation or gene gun transformation, including leaf disc transformation, embryo transformation, and flower bud soaking. Transformed plant cells, tissues, or organs can be regenerated into new plants using conventional methods, thus obtaining transgenic plants.

[0190] In addition, the expression vector preferably contains one or more selective marker genes to provide phenotypic traits for selecting host cells for transformation, such as dihydrofolate reductase, neomycin resistance, and green fluorescent protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance for Escherichia coli.

[0191] Vectors containing the appropriate DNA sequence and appropriate promoter or control sequence can be used to transform appropriate host cells so that they can express proteins.

[0192] The host cell can be a prokaryotic cell, such as a bacterial cell; a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples include: Escherichia coli, Streptomyces; Salmonella typhimurium bacterial cells; fungal cells such as yeast; and plant cells (such as rice cells).

[0193] When the polynucleotides of this invention are expressed in higher eukaryotic cells, the insertion of an enhancer sequence into the vector will enhance transcription. Enhancers are cis-acting factors of DNA, typically approximately 10 to 300 base pairs, that act on the promoter to enhance gene transcription. Examples include the SV40 enhancer (100 to 270 base pairs) located late on the replication origin side, the polyoma enhancer located late on the replication origin side, and adenovirus enhancers.

[0194] Those skilled in the art are well aware of how to select appropriate vectors, promoters, enhancers, and host cells.

[0195] The obtained transformants can be cultured using conventional methods to express the polypeptide encoded by the gene of this invention. Depending on the host cells used, the culture medium can be selected from various conventional media. Culture is carried out under conditions suitable for host cell growth. Once the host cells have grown to an appropriate cell density, the selected promoter is induced using a suitable method (such as temperature adjustment or chemical induction), and the cells are cultured for a further period.

[0196] The proteins described in this invention can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If desired, recombinant proteins can be separated and purified using various separation methods based on their physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitants (salting out), centrifugation, permeation, ultrafiltration, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high-performance liquid chromatography (HPLC), and various other liquid chromatography techniques, as well as combinations of these methods.

[0197] Improve plant traits

[0198] In this invention, a method for improving plant traits is also provided, specifically, the HA1 gene is introduced into plant cells, plant seeds, plant tissues, plant parts or plants to improve plant traits, said traits including (i) promoting the ability of rhizobia to form nodules; (ii) improving nitrogen fixation capacity; and / or (iii) improving quality and yield.

[0199] The main advantages of this invention include:

[0200] (1) This invention is the first to discover that LjHA1 in *Lotus japonicus* is specifically induced by rhizobia; mutations in LjHA1 lead to a reduction in infection events and nodule number. Furthermore, it was found that LjHA1 infection events are further reduced in acidic or alkaline environments compared to normal pH conditions. These results suggest that LjHA1 may participate in the symbiotic nodulation process by regulating pH changes, and further (i) promote the nodulation ability of plant rhizobia; (ii) enhance the nitrogen fixation capacity of plants; and / or (iii) improve the quality and yield of plants.

[0201] (2) This invention discovered H + -ATPase LjHA1 is specifically induced by rhizobia, and mutants of LjHA1 exhibit severe infection events and reduced nodule number. Compared to normal pH environments, infection events in the mutants are further reduced under acidic or alkaline conditions. These results indicate that H +The role of ATPase HA1 in regulating rhizobium infection in host plants in response to changes in external pH is significant, and its function is likely conserved. This study is important for elucidating the molecular mechanism regulating infection line formation and for creating new leguminous materials that can establish effective symbiosis with rhizobia under acidic or alkaline conditions through genetic modification.

[0202] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions as described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise specified, all materials and reagents used in the embodiments are commercially available products.

[0203] Example 1: Expression pattern of LjHA1

[0204] Researchers discovered that Arabidopsis thaliana H + - The ATPase AHA7 has homologous genes in rice (Oryza sativa) and alfalfa, and both OsHA1 and MtHA1 are involved in the regulation of mycorrhizal symbiosis. To investigate H... + Whether ATPase is also involved in the regulation of root nodule symbiosis was investigated by homologous sequence alignment in the Lotus Root Database (https: / / lotus.au.dk / ) and the Alfalfa Database (M.truncatula Mt4.0v1:Phytozome) for all H sequences related to Arabidopsis thaliana. + -Genes homologous to ATPase were identified, and their sequences were analyzed and a phylogenetic tree was constructed. It was found that LotjaGi3g1v0066100 from Lobelia chinensis showed the highest homology with MtHA1, OsHA1, and AtAHA7 from Alfalfa. Therefore, LotjaGi3g1v0066100 was named LjHA1. Figure 1Then, using the root cDNA of *Lotus sibirica* 5 days after inoculation with rhizobia as a template, PCR amplification was performed using primers LjHA1-attBF:GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGGGAGAGGGAAAAGAACCA and LjHA1-attBR:GGGGACCACTTTGTACAAGAAAGCTGGGTCTCAGACAGTGTAGTGCCCGTTCA. A PCR fragment of approximately 3000 bp was obtained. This product was ligated into pDonr207 using the Gateway method to obtain the pDonr207 / LjHA1 vector, and the correctness of the LjHA1 sequence was confirmed by one-step sequencing (SEQ ID NO.1).

[0205] Real-time quantitative PCR was used to determine whether LjHA1 was induced by rhizobia. Roots of wild-type *Gifuloti* sp. inoculated with *Mesorhizobium loti* R7A / lacZ (M. loti R7A / lacZ) rhizobia (obtained from the Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences) at 0, 1, 4, and 14 days were examined to examine the expression level of LjHA1. It was found that LjHA1 was significantly induced by rhizobia. Figure 2 A). Then, we cloned the 2009bp promoter sequence of LjHA1 and constructed the pHA1:GUS expression vector using the Gateway method (Gateway Recombinational Cloning, DOI: 10.1101 / pdb.top094912). This vector was then expressed in wild-type Lophatherum gracile using hairy root transformation (Lotus japonicus Handbook, chapter 6.2 INDUCTION OF HAIRY ROOTS FOR SYMBIOTIC GENE EXPRESSION STUDIES). GUS staining was performed on uninoculated and inoculated roots (14 days after inoculation). The results showed that LjHA1 expression was not observed in uninoculated roots; however, in inoculated roots, LjHA1 was mainly expressed in root nodules and root hairs. Figure 2 B). These results suggest that LjHA1 may play an important role in symbiotic nodules.

[0206] LjHA1 promoter (2009bp):

[0207]

[0208] Example 2: Observation of the symbiotic phenotype of Ljha1

[0209] To investigate the function of LjHA1 in symbiotic nodules, we searched the Lophatherum gracile transposon LORE1 (The LORE1insertion mutant resource, DOI:10.1111 / tpj.13243, a Lophatherum gracile mutant library constructed by researchers at Aarhus University, Denmark through transposon insertion; insertion mutants of the target gene can be ordered for free from the website http: / / lotus.au.dk) and obtained two LjHA1 insertion mutants (ha1-1 and ha1-2). In the ha1-1 mutant, LORE1 is inserted after the 1847th base of the HA1 coding sequence; in the ha1-2 mutant, LORE1 is inserted after the 1421st base of the HA1 coding sequence. Both insertion mutations caused premature termination of HA1 protein translation. We used these two mutants to analyze the symbiotic nodule phenotype. We planted Gifu, ha1-1, and ha1-2 (the mutants were obtained from the Lorelei LORE1 insertion mutant library; we only needed to identify homozygous individuals for propagation and seed collection) in nitrogen-free soil and inoculated them with M. loti R7A / lacZ rhizobium. After 5 days of inoculation, roots were stained with lacZ and infection events were counted. The results showed that compared to the wild type, the number of infection events for ha1-1 and ha1-2 was significantly reduced. Figure 3 AB). Two weeks after inoculation, the number of root nodules was counted, and the results showed that the number of root nodules in ha1-1 and ha1-2 was significantly lower than that in the wild type. Figure 3 CD). This indicates that LjHA1 plays an important role in the symbiotic nodulation process.

[0210] Example 3: Ljha1's ability to adjust to abnormal pH environments is reduced.

[0211] To investigate the ability of LjHA1 to adjust to abnormal pH environments during nodulation and symbiotic root hair infection, we placed Gifu, ha1-1, and ha1-2 plants in normal (pH=6.7), acidic (pH=6), and alkaline (pH=8.5) FP (Fahraeus Medium) media (5mM MgSO4, 7mM KHPO4, 8mM Na2HPO4, 50μM Fe-EDTA, and MnSO4, CuSO4). 4, ZnSO 4, H3BO 3, Five days after inoculation with *M. loli* R7A / lacZ rhizobium (1 μg / L Na2MoO4 each), infection events in the plants were analyzed. Figure 4 ).

[0212] The results showed no difference in infection events among wild-type Gifu under different pH environments; however, the infection events of ha1-1 and ha1-2 were lower than those of wild-type Gifu under normal pH conditions (pH=6.7), and further decreased under acidic (pH=6) or alkaline (pH=8.5) conditions. This indicates that LjHA1 plays an important role in regulating rhizobium infection in host plants in response to changes in external pH.

[0213] in conclusion

[0214] Root hair infection is the primary mode of rhizobium infection selected by most leguminous plants capable of root nodule symbiosis, making the study of this physiological process of great significance. Currently, many mutants with abnormal infection filament development have been reported, but the molecular mechanisms by which host plants regulate infection filament formation in response to changes in the external environment remain unclear. This study found that H... + -ATPase LjHA1 is specifically induced by rhizobia, and mutants of LjHA1 exhibit severe infection events and reduced nodule number. Compared to normal pH environments, infection events in mutants are further reduced under acidic or alkaline conditions. LjHA1 shares more than 80% similarity with MtHA1 in alfalfa, and transcriptome data from alfalfa truncatum show that MtHA1 expression is also induced by rhizobia. These results indicate that H + The role of ATPase HA1 in regulating rhizobium infection in host plants in response to changes in external pH is significant, and its function is likely conserved. This study is important for elucidating the molecular mechanism regulating infection line formation and for creating new leguminous materials that can establish effective symbiosis with rhizobia under acidic or alkaline conditions through genetic modification.

[0215] References

[0216] 1. Jones DT, Taylor WR, Thornton JM. The rapid generation of mutation data matrices from protein sequences. Comput Appl Biosci. 1992; 8(3): 275-282. doi: 10.1093 / bioinformatics / 8.3.275

[0217] 2.Kumar S,Stecher G,Tamura K.MEGA7:Molecular Evolutionary GeneticsAnalysis Version 7.0 for Bigger Datasets.Mol Biol Evol.2016;33(7):1870-1874.doi:10.1093 / molbev / msw054

[0218] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. An isolated polypeptide, characterized in that, The polypeptide is selected from the following group: (a) A polypeptide with the amino acid sequence shown in SEQ ID No. 2; (b) A derivative polypeptide formed by adding a tag sequence, signal sequence or secretion signal sequence to SEQ ID NO.2, and having (i) the ability to promote rhizobium nodulation; (ii) the ability to improve nitrogen fixation; and / or (iii) the activity of improving quality and yield; (c) A derivative polypeptide having an amino acid sequence that is ≥95% homology to the amino acid sequence shown in SEQ ID NO.2, preferably ≥98%, more preferably ≥99%, and having (i) the ability to promote rhizobium nodulation; (ii) the ability to improve nitrogen fixation; and / or (iii) the activity of improving quality and yield.

2. A polynucleotide, characterized in that, The polynucleotide comprises a nucleotide sequence as shown in SEQ ID NO:

1.

3. A carrier, characterized in that, The carrier contains the polynucleotide as described in claim 2.

4. A host cell, characterized in that, The host cell contains the vector of claim 3, or its genome is integrated with the polynucleotide of claim 2.

5. The use of the polypeptide of claim 1, the polynucleotide of claim 2, and the carrier of claim 3, characterized in that, For improving the agronomic traits of plants, or for preparing a composition or formulation for improving the agronomic traits of plants, said agronomic traits including (i) promoting the ability of rhizobia to form nodules; (ii) improving nitrogen fixation capacity; and / or (iii) improving quality and yield.

6. The use of an HA1 gene or its encoded protein, characterized in that, For improving the agronomic traits of plants, or for preparing a composition or formulation for improving the agronomic traits of plants, said agronomic traits including (i) promoting the ability of rhizobia to form nodules; (ii) improving nitrogen fixation capacity; and / or (iii) improving quality and yield.

7. A composition, characterized in that, include: (a) The HA1 gene or its encoded protein; (b) An agriculturally acceptable carrier.

8. Use of the composition according to claim 7, characterized in that, Used to (i) promote the nodulation ability of plant rhizobia; (ii) enhance the nitrogen fixation capacity of plants; and / or (iii) improve plant quality and yield.

9. A method for improving the agronomic traits of plants, characterized in that, The method includes the step of introducing the HA1 gene into plant cells, plant seeds, plant tissues, plant parts, or plants; the improved agronomic traits of the plant include (i) promoting the ability of rhizobia to form nodules; (ii) improving nitrogen fixation capacity; and / or (iii) improving quality and yield.

10. A method for preparing genetically engineered plant tissues or plant cells, characterized in that, Including the following steps: The HA1 gene or its encoded protein is introduced into plant cells or plant tissues to obtain genetically engineered plant tissues or plant cells.

11. A method for preparing plants with improved traits, characterized in that, Including the following steps: The genetically engineered plant tissues or plant cells prepared by the method of claim 10 are regenerated into plant bodies, thereby obtaining plants with improved traits.

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