Use of gmlm1 protein or coding gene thereof and biomaterial containing coding gene thereof in improving lodging resistance and saline-alkali tolerance of plants

Abstract

The present invention relates to the technical field of molecular biology. Provided is the use of a GmLM1 protein or a coding gene thereof and a biomaterial containing the coding gene thereof in improving the lodging resistance and saline-alkali tolerance of plants. Research shows that the GmLM1 protein or the coding gene thereof, the biomaterial containing the coding gene thereof, a mutant of a GmLM1 gene, a biomaterial containing the mutant of the GmLM1 gene, or a biomaterial capable of silencing the GmLM1 gene can regulate the stem strength, stem lignin content, stem cellulose content, leaf total ascorbic acid content and / or saline-alkali tolerance of plants, providing important germplasm resources and theoretical support for cultivating plant varieties with lodging resistance and saline-alkali tolerance. Therefore, the present invention has broad application prospects.

Description

Application of GmLM1 protein or its encoding gene, or biological materials containing its encoding gene in improving plant lodging resistance and salt-alkali resistance Technical Field

[0001] The present invention relates to the technical field of molecular biology, in particular to the application of GmLM1 protein or its coding gene, and biological materials containing the coding gene in improving the lodging resistance and salt-alkali resistance of plants. Background Art

[0002] Soybeans (Glycine max (L.) Merr.) are an annual herbaceous plant in the genus Glycine in the family Leguminosae, widely cultivated worldwide. They are highly nutritious, with protein content several times higher than cereals and tubers. While lower in carbohydrates, soybeans also contain higher levels of essential nutrients such as fat, calcium, phosphorus, iron, and vitamins B1 and B2, making them an ideal, high-quality plant protein source. Eating soybeans and soy products is beneficial to human growth, development, and health. Therefore, soybeans have long been an important food and cash crop.

[0003] Lodging severely impacts soybean yield. Currently, increasing soybean population yield by rationally increasing planting density is a key measure for achieving high soybean yields. Developing superior lodging-resistant soybean varieties is a crucial prerequisite for achieving high and stable yields under dense planting conditions. Therefore, isolating genes that regulate soybean lodging resistance is crucial for increasing soybean yield by enhancing lodging resistance.

[0004] Soil salinization is also a challenge facing agricultural production, and salt stress is a key factor limiting soybean growth and development. Researching and developing salt- and alkali-tolerant soybean varieties can help ensure soybean growth and development, increasing yields and harvests.

[0005] Summary of the Invention

[0006] One of the purposes of the present invention is to provide a new method for changing the lodging resistance and salt-alkali resistance of plants.

[0007] Specifically, the technical solutions of the present invention are as follows:

[0008] In a first aspect, the present invention provides the use of a GmLM1 protein or its encoding gene, a biological material containing its encoding gene, a mutant of the GmLM1 gene, a biological material containing a mutant of the GmLM1 gene, or a biological material that can silence the GmLM1 gene in regulating the stem strength, stem lignin content, stem cellulose content, total ascorbic acid content of leaves and / or salt-alkali tolerance of a plant. The nucleotide sequence of the mutant of the GmLM1 gene is a sequence in which positions 693 to 744 of the CDS sequence of the GmLM1 gene are deleted as shown in SEQ ID NO: 1.

[0009] In a second aspect, the present invention provides the use of a GmLM1 protein or its encoding gene, a biological material containing its encoding gene, a mutant of the GmLM1 gene, a biological material containing a mutant of the GmLM1 gene, or a biological material that can silence the GmLM1 gene in plant breeding or germplasm resource improvement. The nucleotide sequence of the mutant of the GmLM1 gene is a sequence in which positions 693 to 744 of the CDS sequence of the GmLM1 gene are deleted as shown in SEQ ID NO: 1.

[0010] In a third aspect, the present invention provides the use of GmLM1 protein or its encoding gene, or a biological material containing its encoding gene in any of the following aspects:

[0011] (1) Improve plant stem strength;

[0012] (2) Increase the lignin content in plant stems;

[0013] (3) Reduce the cellulose content of plant stems;

[0014] (4) Increase the total ascorbic acid content in plant leaves;

[0015] (5) Improve the salt and alkali tolerance of plants.

[0016] During research, the present invention discovered that mutations in the soybean GmLM1 gene cause the mutant plants to lodging, with reduced stem strength, reduced leaf total ascorbic acid content, reduced lignin content, and increased cellulose content. After functional complementation of the GmLM1 gene in the mutant, the mutant's stem strength, leaf total ascorbic acid content, lignin content, and cellulose content were restored to be essentially the same as those of the wild type. Furthermore, the present invention proposes the use of a protein isolated from soybeans that controls soybean lodging in plant breeding related to lodging traits. Furthermore, overexpression of the GmLM1 gene can improve soybean salt and alkali tolerance, and the GmLM1 protein can also be used to regulate plant salt and alkali tolerance.

[0017] In a fourth aspect, the present invention provides uses of a mutant of the GmLM1 gene, a biomaterial containing the mutant of the GmLM1 gene, or a biomaterial capable of silencing the GmLM1 gene in any of the following aspects:

[0018] (1) Reduce plant stem strength;

[0019] (2) Reduce the lignin content in plant stems;

[0020] (3) Increase the cellulose content of plant stems;

[0021] (4) reduce the total ascorbic acid content in plant leaves;

[0022] (5) Reduce the salt and alkali tolerance of plants;

[0023] The nucleotide sequence of the mutant of the GmLM1 gene is a sequence in which positions 693 to 744 of the CDS sequence of the GmLM1 gene are deleted as shown in SEQ ID NO: 1.

[0024] In the application of the present invention, the amino acid sequence of the GmLM1 protein is one of the following sequences:

[0025] (1) the amino acid sequence shown in SEQ ID NO: 2;

[0026] (2) an amino acid sequence as shown in SEQ ID NO: 2 in which one or more amino acids are substituted, deleted, or added and which has the same or similar function;

[0027] (3) An amino acid sequence that has more than 80% sequence identity (e.g., 80%, 85%, 90%, 95%, 98%, 99% or higher sequence identity) with the amino acid sequence shown in SEQ ID NO: 2 and has the same or similar function.

[0028] In the application of the present invention, the nucleotide sequence of the GmLM1 gene is one of the following sequences:

[0029] (1) the nucleotide sequences shown in SEQ ID Nos: 3-5 connected in sequence;

[0030] (2) A nucleotide sequence encoding a protein having the same or similar function obtained by replacing, deleting or inserting one or more nucleotides in the nucleotide sequence shown in SEQ ID Nos: 3-5 in sequence;

[0031] (3) a nucleotide sequence that can hybridize with the nucleotide sequences shown in SEQ ID Nos: 3-5 linked together under stringent conditions;

[0032] (4) a nucleotide sequence that has a sequence identity of more than 80% with the nucleotide sequences shown in SEQ ID Nos: 3-5, and encodes a nucleotide sequence having the same or similar functional protein;

[0033] (5) A nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID Nos: 3-5 linked in sequence.

[0034] The above-mentioned gene provided in the present invention can be isolated and obtained from plants. Those skilled in the art should know that the GmLM1 gene of the present invention also includes homologous genes that are highly homologous to the nucleotide sequence of the gene and have the same or similar functions after mutation.

[0035] The homologous genes of the present invention specifically have at least 80%, 85%, 90%, 95%, 98%, 99% or higher sequence similarity with the nucleotide sequence of the GmLM1 gene disclosed in the present invention.

[0036] The homologous genes of the present invention also include DNA sequences that can hybridize to the nucleotide sequence of the GmLM1 gene disclosed in the present invention under stringent conditions. The "stringent conditions" of the present invention are conditions well known in the art, for example: (1) a hybridization temperature of 42 degrees Celsius; (2) a hybridization solution of 10% PEG6000; 0.5% SDS; 6×SSC; and 50% formamide.

[0037] In the application of the present invention, the CDS sequence of the GmLM1 gene is shown as SEQ ID No: 1.

[0038] In the application of the present invention, the biological material is an expression cassette, a vector, a host cell or a recombinant bacterium.

[0039] In the application of the present invention, the plant is soybean.

[0040] In a fifth aspect, the present invention provides a DNA molecule, the nucleotide sequence of which is one of the following sequences:

[0041] (1) The sequences shown in SEQ ID Nos: 3-5 are sequentially linked;

[0042] (2) A nucleotide sequence encoding a protein having the same or similar function obtained by replacing, deleting or inserting one or more nucleotides in the nucleotide sequence shown in SEQ ID Nos: 3-5 in sequence;

[0043] (3) a nucleotide sequence that can hybridize with the nucleotide sequences shown in SEQ ID Nos: 3-5 linked together under stringent conditions;

[0044] (4) a nucleotide sequence that has a sequence identity of more than 80% with the nucleotide sequences shown in SEQ ID Nos: 3-5, and encodes a nucleotide sequence having the same or similar functional protein;

[0045] (5) a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID Nos: 3-5 linked in sequence;

[0046] (6) the sequence shown in SEQ ID No: 1;

[0047] (7) The sequence of bases 693 to 744 in SEQ ID NO: 1 is missing.

[0048] In a sixth aspect, the present invention provides a method for constructing transgenic soybeans with improved performance, comprising expressing or overexpressing the GmLM1 gene in soybeans by genetic modification, hybridization, backcrossing, selfing, or asexual propagation; wherein the performance is one or more of stalk strength, stalk lignin content, leaf total ascorbic acid content, and salt-alkali tolerance;

[0049] Preferably, the transgenic method comprises the step of introducing a recombinant expression vector containing the GmLM1 gene into soybean using Ti plasmid, plant virus vector, direct DNA transformation, microinjection, gene gun, electroporation or Agrobacterium-mediated methods.

[0050] In a seventh aspect, the present invention provides a plant mutant, which is based on a wild-type plant and lacks the GmLM1 gene or lacks positions 693 to 744 of the CDS sequence of the GmLM1 gene as shown in SEQ ID NO: 1; preferably, the plant is soybean.

[0051] In an eighth aspect, the present invention provides the use of the above-mentioned plant mutants in regulating plant stem strength, stem lignin content, stem cellulose content, leaf total ascorbic acid content, salt-alkali tolerance, or plant breeding and germplasm resource improvement.

[0052] The beneficial effects of the present invention are at least:

[0053] The present invention provides a new method for improving plant lodging resistance and salt-alkali tolerance, develops a new function of the GmLM1 gene, provides important germplasm resources and theoretical support for breeding lodging-resistant and salt-alkali-tolerant plant varieties, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 shows the results of positional cloning of the gmlm1-1 and gmlm1-2 mutants and a schematic diagram of the GmLM1 gene structure. (a) shows the results of positional cloning of the gmlm1-1 mutant, (b) shows the results of whole-genome resequencing to detect a large deletion in gmlm1-1, (c) shows the results of positional cloning of the gmlm1-2 mutant, and (d) shows the schematic diagram of the GmLM1 gene structure.

[0055] FIG2 is a schematic diagram of the positive expression vector of the soybean GmLM1 gene.

[0056] FIG3 shows the observation results of lodging resistance of T2 soybeans after the gmlm1-1 mutant was transformed with the GmLM1 gene sense plasmid, wild-type soybeans, the gmlm1-1 mutant, and the gmlm1-2 mutant.

[0057] FIG4 shows the statistical results of culm strength of wild-type soybean, gmlm1-1 mutant, gmlm1-2 mutant and gmlm1-1 mutant expressing the GmLM1 gene.

[0058] Figure 5 shows the statistical results of total AsA content (a), cellulose content (b), and lignin content (c) in wild-type soybean, gmlm1-1 mutant, gmlm1-2 mutant, and gmlm1-1 mutant expressing the GmLM1 gene.

[0059] Figure 6 shows the observation results of salt-alkali tolerance of soybean gmlm1-1 mutant overexpressing the GmLM1 gene at the seedling stage.

[0060] Figure 7 shows the expression of the GmLM1 gene in the transcriptomes of wild-type soybean, the gmlm1-1 mutant, the gmlm1-2 mutant, and the gmlm1-1 mutant expressing the GmLM1 gene. In the figure, different lowercase letters on the data bars indicate significant differences in the data. DETAILED DESCRIPTION

[0061] The preferred embodiments of the present invention will be described in detail below with reference to the examples. It should be understood that the following examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention. The following examples are intended to illustrate the present invention but are not intended to limit the scope of the present invention. Unless otherwise specified, the equipment, reagents, and materials used in the examples are all conventionally commercially available.

[0062] The present invention provides the use of the GmLM1 gene or its protein. The nucleic acid molecule encoding the GmLM1 protein includes genomic DNA, cDNA, recombinant DNA or mRNA, hnRNA encoding the GmLM1 protein; or a nucleic acid molecule that is reverse complementary to the above DNA, cDNA, recombinant DNA or mRNA.

[0063] The above nucleic acid molecules can be modified or optimized according to actual needs to make gene expression more efficient; for example, ① based on the codons preferred by the recipient plant, the codons can be changed while maintaining the amino acid sequence of the GmLM1 gene of the present invention to conform to the preference of the recipient plant. ② Modify the gene sequence adjacent to the start methionine to enable efficient translation initiation; for example, modify using a sequence known to be effective in plants. ③ Connect with various plant-expressed promoters to facilitate their expression in plants; the promoters may include constitutive, inducible, temporally regulated, developmentally regulated, chemically regulated, tissue-preferred and tissue-specific promoters; the choice of promoter will vary with the time and space requirements of expression, and also depends on the target species; ④ Introduce enhancer sequences, such as intron sequences (e.g., derived from Adhl and bronzel) and viral leader sequences (e.g., derived from TMV, MCMV and AMV).

[0064] In the present invention, the vector may be a plasmid, cosmid, phage or viral vector. The host may be a fungus, bacteria, algae or cell.

[0065] For plants that do not contain GmLM1, the GmLM1 gene fragment can be introduced into plant cells by chemical methods, shotgun methods, microinjection, electroporation and other methods. The GmLM1 gene fragment can also be introduced into plant cells through homologous recombination, zinc finger nuclease, TALEN, CRISPR and other methods.

[0066] Herein, the words “comprises,” “includes,” and “contains” mean “including but not limited to,” and are not intended to exclude other parts, additives, components, or steps.

[0067] It will be appreciated that features, characteristics, components or steps described in conjunction with a particular aspect, embodiment or example of the invention may be applicable to any other aspect, embodiment or example described herein unless incompatible therewith.

[0068] The present invention will be further described below in conjunction with preferred embodiments:

[0069] Example 1 Research, Isolation and Structural Analysis of the GmLM1 Gene in Soybean

[0070] By subjecting cultivated soybean varieties Williams 82 and Hedou 12 to gamma-ray mutagenesis, the laboratory constructed a large-scale library of gamma-ray-induced mutants. In 2012, the gmlm1-1 mutant with a lodging phenotype was screened from the Hedou 12 background, and in 2014, the gmlm1-2 mutant with a lodging phenotype was screened from the Williams 82 background. Phenotypic characterization from 2014 to 2020 showed that both mutants stably exhibited a lodging phenotype. Hybridization of these two mutants (gmlm1-1 × gmlm1-2) revealed that the resulting F1 hybrid plants exhibited a lodging phenotype similar to that of both parents, indicating that gmlm1-1 and gmlm1-2 are allelic mutants, meaning that the lodging phenotype is caused by mutations in the same gene. To clone the target genes controlling the phenotypes of these two mutants, two F2 genetic segregating populations, gmlm1-1 mutant × Williams82 and gmlm1-2 mutant × Hedou12, were constructed, respectively. Map-based cloning was performed on the two populations, and the results are shown in Figure 1a and c. The target genes were located in the same region of chromosome 2 in both segregating populations. The gmlm1-1 mutant had a sequence deletion in the 20.04Mb-24.90Mb region of chromosome 2 (genome version: Glycine max W82.a2.v1) (see Figure 1b), with a deletion range of 4.86Mb, resulting in the deletion of 44 genes including the GmLM1 gene (Glyma.02G161600-Glyma02G165900). The gmlm1-2 mutant has only one deletion within the GmLM1 gene within the candidate interval, resulting in a gene coding alteration (see Figure 1(d)). This deletion encompasses a total of 1831 bp (positions 7541 to 8330 of the GmLM1 DNA portion as shown in SEQ ID NO:3 and positions 1 to 1041 of the GmLM1 DNA portion as shown in SEQ ID NO:4). This deletion results in the gmlm1-2 mutant encoding a truncated, nonfunctional GmLM1 protein. The amino acid sequence of the GmLM1 protein in gmlm1-mutant 2 is missing amino acids 232-248 of SEQ ID NO:2.

[0071] Isolation of genes:

[0072] mRNA was isolated from young leaves of the cultivated soybean variety Williams 82 and used as a template to generate a 5-mer ... 18(Full-strand gold, AE311) was used as a primer to synthesize the first-strand cDNA. This first-strand cDNA was then used as a template for PCR amplification using primers A (5'-ATGGGAATAGAGATTCTGGAGCCT-3', SEQ ID NO:6) and B (5'-CTAATAAATTCTCTCTGCTGTAGA-3', SEQ ID NO:7). A 1977-bp cDNA fragment of the GmLM1 gene was obtained and cloned into the pGEM T Easy vector (TaKaRa) and designated pGEM T Easy-GmLM1. The CDS sequence of GmLM1 contained in pGEM T Easy-GmLM1 is shown in SEQ ID NO:1, totaling 1977 bp. It encodes a 658-amino acid protein (SEQ ID NO:2, NCBI sequence ID: XP_003518988.1).

[0073] Structural analysis of genes:

[0074] Young leaves of Williams 82 were used as the material to extract DNA from them. Then, the genomic DNA was used as the template to amplify the GmLM1 genome with four pairs of primers: primer C (5'-GTCCATTTCTTTTGTGCTCCG-3', SEQ ID NO:8) and primer D (5'-GGTGGAAGTAAAACGGGTGAAG-3', SEQ ID NO:9), primer E (5'-CTTCACCCGTTTTACTTCCACC-3', SEQ ID NO:10) and primer F (5'-GAATTTAGTTTTGCTACCTTCATGAG-3', SEQ ID NO:11), primer G (5'-CTCATGAAGGTAGCAAAACTAAATTC-3', SEQ ID NO:12) and primer H (5'-GAATTCGGTTGAAGTAATCAGCA-3', SEQ ID NO: 13), primer I (5'-TGCTGATTACTTCAACCGAATTC-3', SEQ ID NO: 14) and primer J (5'-CGGAAAAAGTTTGAAGACGTTGA-3', SEQ ID NO: 15) to obtain the genomic sequence of GmLM1.

[0075] The DNA sequence of GmLM1 is composed of SEQ ID NO:3-5 connected in sequence, totaling 24,886 bp (bp 1-8330 in the DNA sequence of GmLM1 are shown in SEQ ID NO:3, bp 8331-16800 are shown in SEQ ID NO:4, and bp 16801-24886 are shown in SEQ ID NO:5), including 11 introns and 12 exons.

[0076] Among them: 1-324bp is the first exon, of which 1-209bp is 5'UTR; 325-625bp is the first intron; 626-743bp is the second exon; 744-919bp is the second intron; 920-1086bp is the third exon; 1087-1850bp is the third intron; 1851-1918bp is the fourth exon; 1919-2032bp is the fourth intron; 2033-2256bp is the fifth exon; 2257-8278bp is the fifth intron; 8279-8330bp is the sixth exon; 8331-10068bp is the sixth exon 6 introns; 10069-10615bp is the 7th exon; 10616-15637bp is the 7th intron; 15638-15707bp is the 8th exon; 15708-15788bp is the 8th intron; 15789-16008bp is the 9th exon; 16009-16695bp is the 9th intron; 16696-16800bp is the 10th exon; 16801-23904bp is the 10th intron; 23905-24000bp is the 11th exon; 24001-24524bp is the 11th intron; 24525-24886 bp is the 12th exon, of which 24720-24886bp is 3'UTR.

[0077] Example 2 Construction of GmLM1 positive expression vector

[0078] The CDS of GmLM1 (SEQ ID NO: 1) and the CDS of green fluorescent protein (GFP) were fused and forward-linked into the plant expression vector pCAMBIA3301H (CAMBIA Research Center) to obtain a positive-sense expression vector of GmLM1 with a GFP tag. The vector is approximately 11.7 Kbp in length (schematic diagram shown in Figure 2), and the resistance in Escherichia coli is kanamycin resistance, and the resistance in plants is glufosinate resistance.

[0079] Example 3 Agrobacterium-mediated transformation of legumes

[0080] In this example, soybean embryo tips were transformed by Agrobacterium-mediated method to obtain gmlm1-1 mutant explants containing a positive expression vector of the GmLM1 regulatory gene, and their phenotypes were observed, as shown in Figure 3. The specific method is as follows:

[0081] (A) Obtaining soybean explants

[0082] Mature soybean seeds of the gmlm1-1 mutant (GmLM1 gene deletion) with smooth surfaces, no damage, lesions, or cracks were selected and sterilized with chlorine gas for 14 hours. The sterilized seeds were ventilated on a clean bench to completely evaporate the chlorine gas and then germinated in germination medium for 6 hours. Half of the soybean hypocotyl was removed and the soybeans were cut longitudinally along the hypocotyl. The remaining hypocotyl served as the recipient material for Agrobacterium-mediated transformation.

[0083] (B) Soybean transformation

[0084] The vector obtained in Example 2 was introduced into the recipient material using the Agrobacterium-mediated method. Specifically, the vector obtained in Example 2 was transferred into Agrobacterium and sequenced for identification. The Agrobacterium containing the vector obtained in Example 2 was then incubated with the recipient material and cultured in the dark at 22°C on a co-culture medium for 5 days; cultured in SI-I medium under strong light for 7 days; the large buds of the explants were cut off and cultured in SI-II medium under strong light for 14 days; the cotyledons and hypocotyls of the explants were cut off and subcultured every 14 days in SE medium; the approximately 3 cm clustered buds were cut off and placed in rooting medium for rooting; the plants with well-developed roots in RM rooting medium were transferred to soil for planting. Finally, three resistant plants were screened and all three plants tested positive in the Bar test. After five months of culture in the greenhouse, the fruit pods began to mature and the crops were harvested after six months.

[0085] (C) Obtaining heritable transgenic plants and observing their phenotypes

[0086] The seeds of the harvested T1 generation plants, namely the T2 generation, were planted in the field, with 20 plants of each line planted. The leaves at the V2 stage (two tripartite leaves fully expanded) were smeared with herbicide (0.1% v / v) to identify their resistance. Three independent transgenic lines of the gmlm1-1 mutant (GmLM1-OE1, GmLM1-OE2, and GmLM1-OE3) overexpressing the GmLM1 gene were observed at the V8-V9 stage. All lines exhibited an upright growth phenotype consistent with wild-type plants (Williams 82 and Hedou 12), and were taller than the gmlm1-1 mutant (GmLM1 gene deletion), the gmlm1-2 mutant (GmLM1 gene deletions at positions 7541 to 8330 of the partial DNA gene of GmLM1 as shown in SEQ ID NO:3 and positions 1 to 1041 of the partial DNA gene of GmLM1 as shown in SEQ ID NO:4, and positions 693 to 744 of the CDS sequence of the GmLM1 gene as shown in SEQ ID NO:1), and the wild-type plants. The gmlm1-1 and gmlm1-2 mutants exhibited short plant height and curved main stems, indicating lodging.

[0087] Example 4 GmLM1 regulates soybean stalk strength, stalk lignin and cellulose content

[0088] In May 2023, two control varieties (Williams 82 and Hedou 12), two mutants (gmlm1-1 and gmlm1-2), and three independent transgenic lines obtained in Example 3 (GmLM1-OE1, GmLM1-OE2, and GmLM1-OE3) were sown in the experimental field of the Northeast Institute of Geography and Agricultural Ecology, Chinese Academy of Sciences, Changchun City, Jilin Province. At the V7 stage (the seventh trifoliate compound leaf is fully expanded), nodes 1-7 were taken and the stem strength of each node was measured (YYD-1, stem strength tester). The first node of the main stem was dried and ground into powder to determine the cellulose and lignin contents.

[0089] The results showed that the gmlm1-1 and gmlm1-2 mutants had reduced culm strength from nodes 1 to 7 compared to the control varieties Williams 82 and Hedou 12. Three independent transgenic lines (GmLM1-OE1, GmLM1-OE2, and GmLM1-OE3) showed significantly improved culm strength compared to the gmlm1-1 and gmlm1-2 mutants. The specific results are shown in Figure 4.

[0090] Compared with the control varieties Williams 82 and Hedou 12, the gmlm1-1 and gmlm1-2 mutants showed significantly increased cellulose content and significantly decreased lignin content. Three independent transgenic lines (GmLM1-OE1, GmLM1-OE2, and GmLM1-OE3) showed significantly decreased cellulose content and increased lignin content compared with the gmlm1-1 and gmlm1-2 mutants. The results are shown in Figures 5(b) and 5(c).

[0091] Example 5 GmLM1 regulates plant ascorbic acid content

[0092] In July 2023, two control varieties (Williams 82 and Hedou 12), two mutants (gmlm1-1 and gmlm1-2), and three independent transgenic lines obtained in Example 3 (GmLM1-OE1, GmLM1-OE2, and GmLM1-OE3) were sown in an experimental field at the Northeast Institute of Geography and Agroecology, Chinese Academy of Sciences, Changchun, Jilin Province. At the V1 stage (when the first trifoliate leaf was fully expanded), the total ascorbic acid content of the first trifoliate leaf was measured.

[0093] The results showed that the total ascorbic acid content in the leaves of the gmlm1-1 and gmlm1-2 mutants was significantly reduced compared to the control varieties Williams 82 and Hedou 12. Three independent transgenic lines (GmLM1-OE1, GmLM1-OE2, and GmLM1-OE3) showed significantly increased total ascorbic acid content in their leaves compared to the two mutants. This suggests that mutation of the GmLM1 gene significantly reduces ascorbic acid content in plants, while overexpression of the GmLM1 gene can significantly increase ascorbic acid content in plants. The specific results are shown in Figure 5(a).

[0094] Example 6 GmLM1 regulates plant salt-alkali resistance

[0095] Soybean germplasm was cultured in an artificial climate chamber. The culture conditions were a 16 / 8 h light / dark cycle, a temperature of 28°C / 22°C, and a relative humidity of 80%. Uniform, plump, and healthy soybean seeds with intact hilums were selected for planting experiments. The soybean varieties used were the wild-type Williams 82 and the independent transgenic lines GmLM1-OE1 and GmLM1-OE2 obtained in Example 3.

[0096] 1. Salt resistance test:

[0097] 18 pots were planted for each variety, with one seed sown in each pot. The selected seeds were sown in a seedling pot filled with vermiculite for germination. The water absorption holes of the seedling pot were consistent (four water absorption holes). After sowing, the seedling pot was placed in a seedling tray and watered once every three days. When the seedlings grew to the cotyledon stage (VC stage), seedlings with consistent growth were selected for testing, with 6 pots per treatment group. The salt stress treatment group was treated with a NaCl aqueous solution with a concentration of 250mM, and the control treatment group was treated with water (pH=7.0±0.1). Each seedling in the salt stress treatment group was irrigated with 150mL of NaCl aqueous solution (250mM), and each seedling in the control treatment group was irrigated with 150mL of water. On the 3rd day, NaCl aqueous solution / water was applied to the seedlings again with the same dosage. After 7 days, 3 seedlings with consistent growth were selected from the control and salt stress treatment groups of each variety for phenotypic observation and statistics.

[0098] 2. Alkali resistance test:

[0099] Eighteen pots were planted for each variety, with one seed sown in each pot. The selected seeds were sown in a seedling pot filled with vermiculite for germination. The seedling pots had four water absorption holes. After sowing, the seedling pots were placed in a seedling tray and watered every three days. When the seedlings emerged and grew to the cotyledon stage (VC stage), seedlings with uniform growth were selected for the experiment, with 6 pots per treatment group. The alkali stress treatment group was treated with a 150 mmol / L mixed alkali solution (NaHCO3:Na2CO3=5:1; pH=9.0±0.1, 10.50 g of NaHCO3 and 2.65 g of Na2CO3 were weighed to prepare 1 L of 150 mmol / L mixed alkali, and hydrochloric acid and potassium hydroxide were used to adjust the solution pH). The control treatment group was treated with an equal amount of water (pH=7.0±0.1). Each seedling in the alkaline stress treatment group was irrigated with 150 mL of mixed alkaline solution, and each seedling in the control treatment group was irrigated with the same amount of water. After the third day, the same amount of mixed alkaline aqueous solution / water was applied to the seedlings again. After 7 days, three seedlings with consistent growth were selected from each treatment group of each variety for phenotypic observation.

[0100] The specific results are shown in Figure 6. It can be seen that the mutant with the GmLM1 gene complemented has better salt and alkali resistance than the wild-type plant.

[0101] Example 7 Testing of GmLM1 gene expression in soybean transcriptome

[0102] In this example, the expression levels of the GmLM1 gene in wild-type soybeans Williams 82, Hedou 12, the gmlm1-1 mutant, the gmlm1-2 mutant, and the independent transgenic line GmLM1-OE1 obtained in Example 3 were measured (with the internal reference being FPKM). The results are shown in Figure 7. It can be seen that the expression levels of the GmLM1 gene in the mutants with complementation of the GmLM1 gene were higher than those in the wild type.

[0103] Although the present invention has been described in detail above using general descriptions and specific embodiments, it will be apparent to those skilled in the art that modifications and improvements may be made based on the present invention. Therefore, such modifications and improvements, which do not depart from the spirit of the present invention, are intended to be within the scope of protection claimed herein. Industrial Applicability

[0104] The present invention provides the use of a GmLM1 protein or its encoding gene, or a biomaterial containing the encoding gene, for improving plant lodging resistance and salt-alkali resistance. The GmLM1 protein or its encoding gene, biomaterial containing the encoding gene, a GmLM1 gene mutant, a biomaterial containing a GmLM1 gene mutant, or a biomaterial capable of silencing the GmLM1 gene can regulate plant stem strength, stem lignin content, stem cellulose content, leaf total ascorbic acid content, and / or salt-alkali resistance, providing important germplasm resources and theoretical support for the cultivation of lodging-resistant and salt-alkali-tolerant plant varieties and having broad application prospects.

Claims

1. Use of a GmLM1 protein or its encoding gene, a biological material containing its encoding gene, a mutant of the GmLM1 gene, a biological material containing a mutant of the GmLM1 gene, or a biological material capable of silencing the GmLM1 gene in regulating plant stem strength, stem lignin content, stem cellulose content, leaf total ascorbic acid content, and / or salt-alkali tolerance, wherein the nucleotide sequence of the mutant of the GmLM1 gene is a sequence in which positions 693 to 744 of the CDS sequence of the GmLM1 gene as shown in SEQ ID NO: 1 are deleted.

2. Use of a GmLM1 protein or its encoding gene, a biological material containing its encoding gene, a mutant of the GmLM1 gene, a biological material containing a mutant of the GmLM1 gene, or a biological material capable of silencing the GmLM1 gene in plant breeding or germplasm improvement, wherein the nucleotide sequence of the mutant of the GmLM1 gene is a sequence in which positions 693 to 744 of the CDS sequence of the GmLM1 gene as shown in SEQ ID NO: 1 are deleted.

3. Use of the GmLM1 protein or its encoding gene, or a biological material containing the encoding gene, in any of the following aspects: (1) Improve plant stem strength; (2) Increase the lignin content in plant stems; (3) Reduce the cellulose content of plant stems; (4) Increase the total ascorbic acid content in plant leaves; (5) Improve the salt and alkali tolerance of plants.

4. Use of a mutant of the GmLM1 gene, a biological material containing a mutant of the GmLM1 gene, or a biological material capable of silencing the GmLM1 gene in any of the following aspects: (1) Reduce plant stem strength; (2) Reduce the lignin content in plant stems; (3) Increase the cellulose content of plant stems; (4) reduce the total ascorbic acid content in plant leaves; (5) Reduce the salt and alkali tolerance of plants; The nucleotide sequence of the mutant of the GmLM1 gene is a sequence in which positions 693 to 744 of the CDS sequence of the GmLM1 gene are deleted as shown in SEQ ID NO:

1.

5. The use according to any one of claims 1 to 4, characterized in that: The amino acid sequence of the GmLM1 protein is one of the following: (1) the amino acid sequence shown in SEQ ID NO: 2; (2) an amino acid sequence as shown in SEQ ID NO: 2 in which one or more amino acids are substituted, deleted, or added and which has the same or similar function; (3) An amino acid sequence that has a sequence identity of more than 80% with the amino acid sequence shown in SEQ ID NO: 2 and has the same or similar function.

6. The use according to any one of claims 1 to 5, characterized in that: The nucleotide sequence of the GmLM1 gene is one of the following: (1) the nucleotide sequences shown in SEQ ID Nos: 3-5 connected in sequence; (2) A nucleotide sequence encoding a protein having the same or similar function obtained by replacing, deleting or inserting one or more nucleotides in the nucleotide sequence shown in SEQ ID Nos: 3-5 in sequence; (3) a nucleotide sequence that can hybridize with the nucleotide sequences shown in SEQ ID Nos: 3-5 linked together under stringent conditions; (4) a nucleotide sequence that has a sequence identity of more than 80% with the nucleotide sequences shown in SEQ ID Nos: 3-5, and encodes a nucleotide sequence having the same or similar functional protein; (5) A nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID Nos: 3-5 linked in sequence.

7. The use according to any one of claims 1 to 6, characterized in that The CDS sequence of the GmLM1 gene is shown in SEQ ID No:

1.

8. The use according to any one of claims 1 to 7, characterized in that: The biological material is an expression cassette, a vector, a host cell or a recombinant bacterium.

9. The use according to any one of claims 1 to 7, characterized in that: The plant is soybean.

10. A DNA molecule, characterized in that Its nucleotide sequence is one of the following: (1) The sequences shown in SEQ ID Nos: 3-5 are sequentially linked; (2) A nucleotide sequence encoding a protein having the same or similar function obtained by replacing, deleting or inserting one or more nucleotides in the nucleotide sequence shown in SEQ ID Nos: 3-5 in sequence; (3) a nucleotide sequence that can hybridize with the nucleotide sequences shown in SEQ ID Nos: 3-5 linked together under stringent conditions; (4) a nucleotide sequence that has a sequence identity of more than 80% with the nucleotide sequences shown in SEQ ID Nos: 3-5, and encodes a nucleotide sequence having the same or similar functional protein; (5) a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID Nos: 3-5 linked in sequence; (6) the sequence shown in SEQ ID No: 1; (7) The sequence of bases 693 to 744 in SEQ ID NO: 1 is missing.

11. A method for constructing a transgenic soybean with improved performance, characterized in that: The soybean is made to express or overexpress the GmLM1 gene by genetic modification, hybridization, backcrossing, selfing or asexual propagation; the performance is one or more of stalk strength, stalk lignin content, leaf total ascorbic acid content and salt-alkali tolerance; Preferably, the transgenic method comprises the step of introducing a recombinant expression vector containing the GmLM1 gene into soybean using Ti plasmid, plant virus vector, direct DNA transformation, microinjection, gene gun, electroporation or Agrobacterium-mediated methods.

12. A plant mutant, characterized in that: Based on the wild-type plant, the GmLM1 gene is deleted or positions 693 to 744 of the CDS sequence of the GmLM1 gene as shown in SEQ ID NO: 1 are deleted; preferably, the plant is soybean.

13. Use of the plant mutant according to claim 12 in regulating plant stem strength, stem lignin content, stem cellulose content, leaf total ascorbic acid content, salt-alkali tolerance, or in plant breeding and germplasm resource improvement.

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