Method for improving salt tolerance and drought resistance of soybean and application thereof

Abstract

The application belongs to the field of plant breeding, and particularly relates to a method for improving salt tolerance and drought resistance of soybean. The application discloses a gene related to salt tolerance and drought resistance of soybean, and the salt tolerance and drought resistance of soybean can be improved by editing the gene.

Description

Technical Field

[0001] This invention belongs to the field of plant breeding, specifically relating to a method for improving the salt tolerance and drought resistance of soybeans. Background Technology

[0002] Soybeans are one of my country's main food crops and a strategic resource vital to the national economy and people's livelihood. In recent years, human activities have led to frequent natural disasters across the country. Among these, drought and soil salinization have had a significant impact on soybean production, making it crucial to improve the drought and salt tolerance of soybeans.

[0003] Due to the complexity of stress resistance traits, relatively few genes related to stress resistance have been identified in soybeans, which to some extent limits the technical means that can be used to breed new stress-resistant soybean varieties. Summary of the Invention

[0004] To address the aforementioned problems, the present invention aims to disclose a protein and nucleic acid sequence related to soybean drought resistance and salt tolerance, provide a method for improving soybean salt tolerance and / or drought resistance, and provide artificially mutated genes and mutant proteins with high salt tolerance and / or drought resistance effects.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] This invention provides an application of a soybean gene in improving the salt tolerance and / or drought resistance of soybeans, characterized in that the sequence of the gene is shown in SEQ ID NO.2 or SEQ ID NO.3.

[0007] The present invention also provides a method for improving the salt tolerance and / or drought resistance of soybeans, characterized in that the expression and / or activity of the above-mentioned genes are reduced in the soybean material to be improved, and plants with increased salt tolerance and / or drought resistance are selected.

[0008] In some implementations, the methods for reducing protein expression and / or activity include any one of gene editing, RNA interference, or site-directed insertion of an inhibitory element.

[0009] In some implementations, the target sequence for the gene editing described above is shown in SEQ ID NO.4.

[0010] In some implementations, the gene editing described above uses the CRISPR / Cas9 method.

[0011] This invention also provides a kit for improving the salt tolerance and / or drought resistance of soybeans, characterized in that it comprises any one of the following 1) to 3) and Cas9 protein:

[0012] (1) An RNA molecule capable of recognizing the above target sequence; in some embodiments, the sequence of the above RNA molecule is shown in SEQ ID NO.5;

[0013] (2) The DNA molecule encoding the RNA described in (1);

[0014] (3) Vectors that express the RNA described in (1).

[0015] The present invention also provides a mutant gene, characterized in that: the sequence of the mutant gene is shown in SEQ ID NO.6.

[0016] The present invention also provides a mutant protein, characterized in that: the amino acid sequence of the mutant protein is shown in SEQ ID NO. 8.

[0017] This invention also provides a method for breeding soybeans, characterized in that the method includes the following steps:

[0018] 1) Soybeans from which the above-mentioned mutated genes were obtained;

[0019] 2) The soybeans obtained in step 1) are subjected to pollen culture, unfertilized embryo culture, doubling culture, cell culture, tissue culture, self-pollination or hybridization or a combination thereof to obtain soybean plants, seeds, plant cells, progeny plants or plant parts;

[0020] 3) The offspring soybean plants obtained in step 2) are subjected to drought resistance or salt tolerance assessment, and soybean plants with improved salt tolerance and / or drought resistance are selected.

[0021] The beneficial effects of this invention are as follows: Through gene screening and stress resistance identification, this invention obtained a gene, GmTRE, related to soybean salt tolerance and drought resistance, and clearly demonstrated that editing this gene can improve soybean salt tolerance and drought resistance. This invention also identified a mutant gene that can result in superior performance in both stress resistance and yield traits in soybeans. All of the above-mentioned inventions contribute to the breeding of new salt-tolerant and drought-resistant soybean varieties. Attached Figure Description

[0022] Figure 1 pGWB535 carrier diagram. Detailed Implementation

[0023] The following definitions and methods are provided to better define this application and to guide those skilled in the art in its practice. Unless otherwise stated, the terms are to be understood in accordance with their conventional usage by those skilled in the art. All patent literature, academic papers, industry standards, and other publicly available publications cited herein are incorporated herein by reference in their entirety.

[0024] In this application, the words “comprising,” “including,” or variations thereof should be understood to include other elements, numbers, or steps in addition to those described.

[0025] Unless otherwise specified, nucleic acids are written from left to right in a 5' to 3' orientation; amino acid sequences are written from left to right in an amino-to-carboxyl orientation. Amino acids may be represented herein by their commonly known three-letter symbols or by the single-letter symbols recommended by the IUPAC-IUB Committee on Biochemistry Nomenclature. Similarly, nucleotides may be represented by commonly accepted single-letter codes. Numerical ranges include numbers that define the range. As used herein, “nucleic acid” includes deoxyribonucleotides or ribonucleotide polymers in single-stranded or double-stranded form, and unless otherwise limited, includes known analogs (e.g., peptide nucleic acids) that have the basic properties of natural nucleotides and hybridize with single-stranded nucleic acids in a manner similar to naturally occurring nucleotides. As used herein, the terms “encoding” or “encoded” when used in the context of a particular nucleic acid mean that the nucleic acid contains the essential information to guide the translation of that nucleotide sequence into a particular protein. Codons are used to represent the information encoding the protein. As used herein, “full-length sequence” referring to a particular polynucleotide or the protein it encodes means the entire nucleic acid sequence or the entire amino acid sequence having a natural (non-synthetic) endogenous sequence. Full-length polynucleotides encode the full-length, catalytically active form of the specific protein. The terms “peptide,” “polypeptide,” and “protein” are used interchangeably herein to refer to polymers of amino acid residues. This term is used for amino acid polymers in which one or more amino acid residues are artificial chemical analogs of the corresponding naturally occurring amino acids. This term is also used for naturally occurring amino acid polymers. The terms “residue,” “amino acid residue,” or “amino acid” are used interchangeably herein to refer to an amino acid incorporated into a protein, polypeptide, or peptide (collectively, “protein”). Amino acids can be naturally occurring amino acids and, unless otherwise limited, may include known analogs of natural amino acids that can function in a similar manner to naturally occurring amino acids.

[0026] In some embodiments, the nucleotide sequence of this application may be modified to perform conserved amino acid substitutions. Principles and examples of conserved amino acid substitutions are further described below. In some embodiments, the nucleotide sequence of this application may be substituted without altering the amino acid sequence according to disclosed monocotyledonous codon preferences; for example, a codon encoding the same amino acid sequence may be substituted with a codon preferred by monocotyledons without changing the amino acid sequence encoded by the nucleotide sequence. In some embodiments, a portion of the nucleotide sequence in this application may be substituted with a different codon encoding the same amino acid sequence, thereby changing the nucleotide sequence without altering the encoded amino acid sequence. Conserved variants include those sequences that encode an amino acid sequence of one of the proteins of the embodiments due to genetic codon degeneracy. In some embodiments, a portion of the nucleotide sequence in this application may be substituted according to a codon preferred by monocotyledons. Those skilled in the art will recognize that amino acid additions and / or substitutions are generally based on the relative similarity of amino acid side-chain substituents, such as the hydrophobicity, charge, size, etc., of the substituents. Exemplary amino acid substituents having the various properties considered above are well known to those skilled in the art and include arginine and lysine; glutamic acid and aspartic acid; serine and threonine; glutamine and asparagine; and valine, leucine, and isoleucine. Guidance on appropriate amino acid substitutions that do not affect the biological activity of the target protein can be found in the model of Dayhoff et al. (1978) Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, DC) (incorporated herein by reference). Conserved substitutions, such as replacing one amino acid with another amino acid having similar properties, can be performed. Sequence identity verification includes hybridization techniques. For example, a known nucleotide sequence, in whole or in part, can be used as a probe for selective hybridization with other corresponding nucleotide sequences present in cloned genomic DNA fragments or cDNA fragment groups (i.e., genomic libraries or cDNA libraries) from a selected organism. The hybridization probe may be a genomic DNA fragment, cDNA fragment, RNA fragment, or other oligonucleotide, and may be labeled with a detectable group such as 32P or other detectable markers. Thus, for example, hybridization probes can be prepared by labeling synthetic oligonucleotides based on sequences from the embodiment. Methods for preparing hybridization probes and constructing cDNA and genomic libraries are generally known in the art. Hybridization of the sequences can be performed under stringent conditions. As used herein, the terms "stringent conditions" or "stringent hybridization conditions" refer to conditions under which the probe will hybridize with its target sequence to a detectable extent (e.g., at least 2, 5, or 10 times the background) relative to hybridization with other sequences.Harsh conditions are sequence-dependent and vary across different environments. By controlling hybridization harshness and / or washing conditions, target sequences 100% complementary to the probe can be identified (homologous probe method). Alternatively, harsh conditions can be adjusted to allow for some sequence mismatches in order to detect lower similarities (heterologous probe method). Typically, probe lengths are less than about 1000 or 500 nucleotides. Typically, harsh conditions are those where the salt concentration is less than about 1.5 M Na ions at pH 7.0 to 8.3, typically about 0.01 M to 1.0 M Na ion concentration (or other salts), and the temperature conditions are: at least about 30 °C for short probes (e.g., 10 to 50 nucleotides) and at least about 60 °C for long probes (e.g., greater than 50 nucleotides). Harsh conditions can also be achieved by adding a destabilizing agent such as formamide. Exemplary low-threshold conditions include hybridization at 37°C using 30% to 35% formamide buffer, 1M NaCl, and 1% SDS (sodium dodecyl sulfate), followed by washing at 50°C to 55°C in 1× to 2× SSC (20× SSC = 3.0M NaCl / 0.3M trisodium citrate). Exemplary medium-threshold conditions include hybridization at 37°C using 40% to 45% formamide, 1.0M NaCl, and 1% SDS, followed by washing at 55°C to 60°C in 0.5× to 1× SSC. Exemplary high-threshold conditions include hybridization at 37°C using 50% formamide, 1M NaCl, and 1% SDS, followed by a final wash at 60°C to 65°C in 0.1× SSC for at least about 20 minutes. Optionally, the wash buffer may contain about 0.1% to about 1% SDS. Hybridization duration is typically less than about 24 hours, typically from about 4 hours to about 12 hours. Specificity typically depends on post-hybridization washing, with key factors being the ionic strength and temperature of the final washing solution. The Tm (thermodynamic melting point) of DNA-DNA hybrids can be approximated by the formula from Meinkoth and Wahl (1984) Anal. Biochem. 138:267-284: Tm = 81.5℃ + 16.6 (logM) + 0.41 (%GC) - 0.61 (%formamide) - 500 / L; where M is the molar concentration of monovalent cations, %GC is the percentage of guanosine and cytosine nucleotides in the DNA, "formamide%" is the percentage of formamide in the hybridization solution, and L is the base pair length of the hybrid. Tm is the temperature at which 50% of the complementary target sequence hybridizes with a perfectly matched probe (at a given ionic strength and pH). Washing is typically performed at least until equilibration is reached and a low hybridization background level is achieved, such as for 2 hours, 1 hour, or 30 minutes. Each 1% mispairing should lower Tm by approximately 1°C; therefore, Tm, hybridization, and / or washing conditions can be adjusted to hybridize with the desired sequence of homology. For example, if a sequence with ≥90% homology is required, Tm can be lowered by 10°C.Typically, the stringency conditions are selected to be approximately 5°C lower than the Tm of the specific sequence and its complementary sequence at the defined ionic strength and pH. However, under very stringent conditions, hybridization and / or washing can be performed at 4°C lower than the Tm; under moderately stringent conditions, hybridization and / or washing can be performed at 6°C lower than the Tm; and under low stringency conditions, hybridization and / or washing can be performed at 11°C lower than the Tm.

[0027] Unless otherwise specified, all figures representing amounts of components, reaction conditions, etc., used in this specification and claims should be understood to be modified by the term "about" in all cases. As used herein, the term "about," when referring to a measurable value such as mass, weight, time, volume, concentration, or percentage, means to cover variations of ±20% from a specified amount in some embodiments, ±10% in some embodiments, ±5% in some embodiments, ±1% in some embodiments, ±0.5% in some embodiments, and ±0.1% in some embodiments, because such variations are suitable for performing the disclosed methods and / or using the disclosed compositions, nucleic acids, peptides, etc. Therefore, unless indicated to the contrary, the numerical parameters listed in this specification and appended claims are approximate values ​​that may vary depending on the desired characteristics sought to be obtained from the subject matter disclosed in this application.

[0028] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and substance thereof are within the scope of this application. Unless otherwise specified, the examples are conducted under conventional experimental conditions, such as those described in Sambrook et al.'s Molecular Cloning Laboratory Manual (Sambrook J & Russell DW, Molecular cloning: alaboratory manual, 2001), or according to the conditions recommended in the manufacturer's instructions. Unless otherwise specified, the chemical reagents used in the examples are all commercially available conventional reagents, and the technical means used in the examples are conventional means well known to those skilled in the art.

[0029] Example

[0030] Example 1: Discovery of genes related to salt tolerance and drought resistance in soybeans

[0031] Trehalose can be catalyzed by trehalase to produce glycosides, and trehalase plays a role in abiotic stress tolerance. However, it remains unclear which gene in soybean is involved in trehalose metabolism, which type of abiotic stress it is associated with, and how to manipulate it to improve soybean's tolerance to abiotic stress.

[0032] The inventors first used bioinformatics methods to analyze and identify several genes related to the trehalose metabolism pathway, including five trehalose C6 phosphate synthase genes, two trehalose-6 phosphate esterase genes, and one trehalase gene. Therefore, editing a trehalase gene could regulate trehalose metabolism. They then attempted to mutate these genes using gene editing methods to test their effects on tolerance to abiotic stress.

[0033] The gene editing vector backbone is the soybean gene editing vector pGES401 (see Bai M, Yuan C, Kuang H, et al. Combination of two multiplex genome-edited soybean varieties enable customization of protein functional properties[J]. Mol Plant. 2022, 15(7):1081-1083.). The sgRNA target sequence can be designed using online tools, such as http: / / crispr.dbcls.jp / . After Oligo annealing, it is cloned into the pGES401 vector by Bsa I restriction enzyme digestion.

[0034] The constructed soybean gene-editing vector was used for soybean genetic transformation via Agrobacterium-mediated cotyledon node infection. Soybean seeds were first sterilized with chlorine gas, and after germination on a culture medium, cotyledon nodes were collected as explants and infected with Agrobacterium. The explants then underwent induction culture, elongation culture, and rooting culture for selection. Basta resistance was selected. T0 resistant seedlings were preliminarily identified by PCR and then transferred to soil culture for seed harvesting. After T1 generation seed germination, Basta was applied to the leaves, and positive plants were further identified by PCR to obtain stable transformed plants for subsequent experiments.

[0035] The obtained edited plants were subjected to salt and drought stress treatments, specifically as follows:

[0036] (1) Vermiculite culture salt stress treatment:

[0037] Seven days after sowing, healthy and uniform soybean seedlings were selected and transplanted into new small black boxes, each containing the same volume of vermiculite. Six seedlings were transplanted into each small black box for further cultivation. Salt stress treatment was initiated 10 days after sowing. The stock solution of low-nitrogen soybean nutrient solution was diluted and sodium chloride solid was added to prepare a 250 mmol / L salt stress treatment solution, which was then adjusted to pH 5.8-6.0. 150 mL of the salt stress treatment solution was added to each black box in the treatment group each time, while 150 mL of the low-nitrogen soybean nutrient solution was added to each control group each time. Plant survival rates were recorded after a clear phenotype was observed following salt treatment.

[0038] (2) Vermiculite culture under drought stress treatment:

[0039] Seven days after sowing, healthy and uniform soybean seedlings were selected and transplanted into new small black boxes, each containing the same volume of vermiculite. Six seedlings were transplanted into each box for further cultivation. In the drought stress treatment, the treatment group and the control group were watered with the same volume of low-nitrogen soybean nutrient solution 10 days after sowing, after which watering was withheld. The control group received additional low-nitrogen soybean nutrient solution during the treatment period. Different lines underwent rehydration treatment after exhibiting distinct phenotypes during drought treatment. Plant survival rates were recorded on the fifth day after rehydration.

[0040] The results showed that the two gene-edited plants exhibited greater tolerance to salt and drought stress. Further studies revealed that the H2O2 content in these two gene-edited plants was also significantly reduced.

[0041] The identification and analysis of the editing target showed that the target sequence of both gene-edited plants was "5'-TTTTGGATGCTCAAGGTTGC-3'", which is located in the exon region of the soybean GmTRE gene. The genomic sequence of the GmTRE gene is shown in SEQ ID NO.3, the coding region sequence is shown in SEQ ID NO.2, and the amino acid sequence is shown in SEQ ID NO.1.

[0042] The results showed that the GmTRE gene in both edited plants was truncated after editing, resulting in a knockout effect. The coding region sequences of the edited GmTRE gene in the two edited plants are shown in SEQ ID NO.6 and SEQ ID NO.8, and the amino acid sequences are shown in SEQ ID NO.7 and SEQ ID NO.9.

[0043] The inventors further constructed soybean plants overexpressing the GmTRE gene (overexpression vector used was pGWB602, vector diagram shown). Figure 1For specific information, please refer to Nakagawa T, Suzuki T, Murata S, et al. Improved Gatewaybinary vectors: high-performance vectors for creation of fusion constructs in transgenic analysis of plants. Biosci Biotechnol Biochem., 2007, 71(8): 2095-100. The phenotypic identification results showed that, compared with WT, the GmTRE overexpression line exhibited sensitivity to salt and drought stress, higher H2O2 content, and lower survival rate. These results indicate that GmTRE has a negative regulatory effect on salt tolerance and drought resistance.

[0044] Table 2. Phenotypic Performance of Plants Edited with GmTRE Gene

[0045]

[0046] Example 2: Screening of Superior Plants

[0047] The inventors further investigated the agronomic traits of the two GmTRE gene-edited lines under normal field cultivation conditions, including plant height, number of branches, number of pods, 100-seed weight, single-plant seed weight, protein content, and oil content. The results showed that although the survival rate of the gmtre-2 line under salt and drought stress was higher than that of the gmtre-1 line, and its plant height was also greater (closer to the recipient control), its seed number and seed weight were the lowest. Therefore, considering both stress resistance and yield performance, gmtre-1 is the superior edited line. The gmtre-1 line can be used to breed new salt-tolerant or drought-resistant soybean varieties (for example, by introducing the GmTRE mutant gene from gmtre-1 into other soybean germplasm through backcrossing, or by using genetic engineering to modify the GmTRE gene in other soybean germplasm into the mutant gene in gmtre-1).

[0048] Table 2. Phenotypic Performance of Plants Edited with GmTRE Gene

[0049]

[0050] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. The application of reducing soybean gene expression in improving soybean salt tolerance and / or drought resistance, characterized in that, The genome sequence of the gene is shown in SEQ ID NO. 3, and the coding region sequence is shown in SEQ ID NO.

2.

2. A method for improving the salt tolerance and / or drought resistance of soybeans, characterized in that, Reduce the expression of the gene described in claim 1 in the soybean material to be improved, and select plants with increased salt tolerance and / or drought resistance.

3. The method according to claim 2, characterized in that: The methods for reducing gene expression include any one of gene editing, RNA interference, or site-directed insertion of an inhibitory element.

4. The method according to claim 3, characterized in that: The target sequence for gene editing is shown in SEQ ID NO.

4.

5. The method according to claim 3, characterized in that: The gene editing was performed using the CRISPR / Cas9 method.

6. The application of a reagent kit in improving the salt tolerance and / or drought resistance of soybeans, characterized in that: The kit includes any one of the following 1) to 3) and Cas9 protein: (1) An RNA molecule capable of recognizing the target sequence described in claim 4; (2) The DNA molecule encoding the RNA described in (1); (3) A vector for expressing the RNA described in (1).

7. The application according to claim 6, characterized in that: The RNA molecule sequence is shown in SEQ ID NO. 5.

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