Soybean transcription factor GmERF416 and application of coding gene thereof in regulation and control of plant salt tolerance

Abstract

The invention discloses application of a soybean transcription factor GmERF416 and a coding gene thereof in regulation and control of plant salt tolerance. The invention relates to the technical field of biology, in particular to application of a soybean transcription factor GmERF416 and a coding gene thereof in regulation and control of plant salt tolerance. The protein disclosed by the invention is a protein with an amino acid sequence of SEQ ID No.2, and can be applied to: 1) regulating and controlling the salt tolerance of plants; 2) preparing a product for regulating and controlling the salt tolerance of the plants; (3) cultivating plants with changed salt resistance; 4) preparing a product for cultivating the plant with changed salt resistance; and 5) plant breeding. By knocking out the GmERF416 protein, the salt tolerance of soybeans can be improved, and the method has guiding significance for breeding high-quality soybean varieties.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to the application of soybean transcription factor GmERF416 and its encoding gene in regulating plant salt tolerance. Background Technology

[0002] Changes in physical and chemical factors in the environment, such as drought, salinity, cold damage, frost damage, and waterlogging, are among the causes of severe crop yield reductions. Statistics from insurance payouts in the United States between 1939 and 1978 show that approximately 40.8% of payouts were due to salinity and drought, higher than waterlogging (16.4%), low temperature (13.8%), hail (11.3%), and wind (7.0%), and far higher than insect infestations (4.5%), diseases (2.7%), and other factors. Utilizing low- to moderately saline land for soybean, corn, and rice production, and cultivating salt- and drought-tolerant crops, has become a major goal in agriculture. Besides traditional breeding methods, molecular genetic breeding has become a focus of scientific research in improving crop salt and drought tolerance.

[0003] Soybeans, as an important economic crop, are a significant source of edible oils and plant protein, holding a vital position in my country's food industry. Soybean oil also has numerous applications in industrial production, such as biofuels, surfactants, and plasticizers. In recent years, my country's soybean production has only accounted for one-fifth of its demand, making China the world's largest soybean importer. This falls far short of meeting national needs. Highly salt-tolerant soybeans can maximize the utilization of saline-alkali soils, representing one strategy for increasing my country's soybean yield.

[0004] GmERF416 belongs to the AP2 / ERF family of transcription factors found only in plants. The AP2 / ERF family is a large family, named for its AP2 / ERF domain, which consists of 60-70 amino acids. They participate in a variety of biological processes, including plant growth, flower, fruit and seed development, damage repair, pathogen defense, and responses to abiotic stresses. AP2 / ERF transcription factors are involved in multiple hormone signaling pathways, including those involving salicylic acid, jasmonic acid, ethylene, and abscisic acid. Summary of the Invention

[0005] The technical problem to be solved by this invention is how to improve the salt tolerance of plants.

[0006] To address the aforementioned problems, the present invention provides substances that regulate the expression of protein-coding genes or regulate the content of said proteins in any of the following applications:

[0007] A1) Applications in improving plant salt tolerance / or in the preparation of products that improve plant salt tolerance;

[0008] A2) Applications in the breeding of plants with high salt tolerance and / or the preparation of products with high salt tolerance;

[0009] The protein may be any of the following:

[0010] B1) The amino acid sequence is that of the protein shown in SEQ ID No. 2;

[0011] B2) A protein obtained by substituting and / or deleting and / or adding amino acid residues of the protein described in B1), which has more than 80% identity with the protein shown in B1) and has the function of regulating plant salt tolerance.

[0012] B3) A fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of B1) or B2).

[0013] In the aforementioned proteins, the protein tag refers to a polypeptide or protein fused with the target protein using in vitro DNA recombination technology for expression, detection, tracing, and / or purification of the target protein. The protein tag may be a Flag tag, His tag, MBP tag, HA tag, myc tag, GST tag, and / or SUMO tag, etc.

[0014] Of the proteins mentioned above, SEQ ID No. 2 consists of 416 amino acid residues and is named GmERF416 protein or protein GmERF416. Its encoding gene is the GmERF416 gene.

[0015] In this article, the substance that regulates the activity and / or content of the protein may be a substance that regulates gene expression, wherein the gene encodes the protein GmERF416.

[0016] In the above applications, the protein is derived from soybean (Glycine max(L.)Merr.).

[0017] In the above text, the substance regulating gene expression can be a substance that performs at least one of the following six types of regulation: 1) regulation at the transcriptional level of the gene; 2) post-transcriptional regulation of the gene (i.e., regulation of splicing or processing of the primary transcript of the gene); 3) regulation of RNA transport of the gene (i.e., regulation of mRNA transport of the gene from the nucleus to the cytoplasm); 4) regulation of translation of the gene; 5) regulation of mRNA degradation of the gene; and 6) post-translational regulation of the gene (i.e., regulation of the activity of the protein translated from the gene).

[0018] In the above applications, the substance regulating the expression of the gene encoding the protein is any one of the following:

[0019] c1) The nucleic acid molecule that encodes the protein described above;

[0020] c2) An expression cassette containing the nucleic acid molecule described in c1);

[0021] c3) A recombinant vector containing the nucleic acid molecule described in c1), or a recombinant vector containing the expression cassette described in c2);

[0022] c4) Recombinant microorganisms containing the nucleic acid molecules described in c1), or recombinant microorganisms containing the expression cassette described in c2), or recombinant microorganisms containing the recombinant vector described in c3);

[0023] c5) A transgenic plant cell line containing the nucleic acid molecule described in c1), or a transgenic plant cell line containing the expression cassette described in c2);

[0024] c6) Transgenic plant tissue containing the nucleic acid molecules described in c1), or transgenic plant tissue containing the expression cassette described in c2);

[0025] c7) A transgenic plant organ containing the nucleic acid molecule described in c1), or a transgenic plant organ containing the expression cassette described in c2);

[0026] e1) Nucleic acid molecules that inhibit, reduce, or silence the expression of the protein-encoding genes mentioned above;

[0027] e2) An expression cassette containing the nucleic acid molecule described in e1);

[0028] e3) A recombinant vector containing the nucleic acid molecule described in e1), or a recombinant vector containing the expression cassette described in e2);

[0029] e4) Recombinant microorganisms containing the nucleic acid molecules described in e1), or recombinant microorganisms containing the expression cassette described in e2), or recombinant microorganisms containing the recombinant vector described in e3);

[0030] e5) A transgenic plant cell line containing the nucleic acid molecule described in e1), or a transgenic plant cell line containing the expression cassette described in e2);

[0031] e6) Transgenic plant tissue containing the nucleic acid molecules described in e1), or transgenic plant tissue containing the expression cassette described in e2);

[0032] e7) A transgenic plant organ containing the nucleic acid molecule described in e1) or a transgenic plant organ containing the expression cassette described in e2).

[0033] Those skilled in the art can readily mutate the nucleotide sequence encoding the GmERF416 protein gene that inhibits, reduces, or downregulates its expression using known methods, such as directed evolution or point mutation. Artificially modified nucleotides that possess 80% or more of the same nucleotide sequence as the GmERF416 protein gene encoding sequence isolated according to this invention, and which also have the function of inhibiting, reducing, or downregulating the expression of the GmERF416 protein gene, are all derived from and equivalent to the nucleotide sequence of this invention.

[0034] In this article, identity refers to the similarity of amino acid or nucleotide sequences. The identity of amino acid sequences can be determined using homology search sites on the internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST 2.1, using blastp as the procedure, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as the matrix, setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values) respectively, and performing a search to calculate the identity of amino acid sequences, then the identity value (%) can be obtained.

[0035] In this document, the 80% or more identity can be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

[0036] The nucleic acid molecules mentioned in this article can be DNA, such as cDNA, genomic DNA, or recombinant DNA; the nucleic acid molecules can also be RNA, such as gRNA, mRNA, siRNA, shRNA, sgRNA, miRNA, or antisense RNA.

[0037] In the above applications, the nucleic acid molecule described in c1) can be any of the following DNA molecules:

[0038] d1) The nucleotide sequence is the DNA molecule shown in SEQ ID No. 3;

[0039] d2) The coding region sequence is the DNA molecule shown in SEQ ID No. 1;

[0040] d3) has 90% or more identity with the nucleotide sequence defined by d1) or d2) and is a DNA molecule encoding the protein described above;

[0041] d4) Hybridizes under strict conditions to a nucleotide sequence defined by d1) or d2) and encodes a DNA molecule that encodes the protein described above.

[0042] In the above application, the expression cassette described in e2) contains a DNA molecule with the nucleotide sequence shown in SEQ ID No. 4.

[0043] In the above applications, e1) the nucleic acid molecule is a gRNA that targets the protein-coding gene mentioned above, and the target sequence of the gRNA may be 5'-TCTCGCAGCTCGCAGTACCG-3' or / and 5'-ACTCCCATCTACCAGTCCTG-3'.

[0044] The vectors described herein are known to those skilled in the art and include, but are not limited to: plasmids, bacteriophages (such as λ phage or M13 filamentous phage), granules (i.e., Cos plasmids), Ti plasmids, or viral vectors.

[0045] In the above e3), the recombinant vector can be a plant gene editing vector. The plant gene editing vector can be a knockout vector pCBSG015-sgRNA. In this invention, the pCBSG015-sgRNA vector contains the AtU6 promoter and carries the spectinomycin gene.

[0046] In this invention, the knockout is performed using a dual-gene, dual-target knockout method.

[0047] The recombinant vector pCBSG015-sgRNA is a recombinant plasmid obtained by inserting DNA fragments containing the sequences 5'-TCTCGCAGCTCGCAGTACCG-3' (sgRNA1) and 5'-ACTCCCATCTACCAGTCCTG-3' (sgRNA2) between the restriction endonuclease Bsa I sites of the pCBSG015 vector, while keeping other sequences of the pCBSG015 vector unchanged.

[0048] The target sequence of sgRNA1 is the DNA fragment at positions 472-491 of sequence 1 or the DNA fragment at positions 472-491 of sequence 3, and the target sequence of sgRNA2 is the DNA fragment at positions 510-529 of sequence 1 or the DNA fragment at positions 510-529 of sequence 3.

[0049] The microorganism mentioned in (e4) above can be Agrobacterium. The Agrobacterium is EHA105.

[0050] This invention also provides a method for regulating the salt tolerance of plants.

[0051] The method for regulating plant salt tolerance provided by the present invention includes regulating plant salt tolerance by regulating the expression of the gene encoding the protein GmERF416, or regulating the activity and / or content of the protein, or regulating the activity and / or content of the gene encoding the protein.

[0052] In this invention, the regulation can be increased, enhanced, or raised. The regulation can also be decreased, weakened, or reduced.

[0053] In this article, the enhancement, increase or upregulation of the expression level of the coding gene of the aforementioned protein in the recipient plant, and / or the enhancement, increase or upregulation of the activity and / or content of the coding gene of the aforementioned protein, is achieved by introducing the coding gene of the aforementioned protein into the recipient plant.

[0054] In this article, regulating the expression of the gene encoding the protein can be achieved by inhibiting, reducing, or downregulating the expression of the gene. Inhibition, reduction, or downregulation of the gene expression can be achieved through gene knockout or gene silencing.

[0055] Gene knockout refers to the phenomenon of inactivating a specific target gene through gene editing technology. Gene knockout inactivates a specific target gene by altering its DNA sequence, including but not limited to zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the CRISPR / Cas system. CRISPR (clustered regulatory interspaced short palindromic repeat) is a site in the genome containing multiple short repeat sequences, where the Cas9 protein, mediated by RNA, can cleave target sequences recognized by crRNA–tracrRNA.

[0056] This invention also provides a method for cultivating plants with altered salt tolerance.

[0057] The present invention provides a method for cultivating plants with improved salt tolerance, comprising inhibiting or reducing or silencing the expression of the coding gene of the above-mentioned protein and / or the content and / or activity of the above-mentioned protein in the target plant, or / and inhibiting or reducing or silencing the activity and / or content of the coding gene of the above-mentioned protein, thereby obtaining plants with improved salt tolerance.

[0058] In one embodiment of the present invention, the breeding method for cultivating plants with improved salt tolerance includes the following steps:

[0059] (1) Construct recombinant expression vectors that suppress, reduce, or silence the proteins described above;

[0060] (2) The recombinant expression vector constructed in step (1) is transferred into the recipient plant to obtain a plant with higher salt tolerance than the recipient plant.

[0061] In this invention, the purpose of plant breeding may include cultivating plants with improved salt tolerance.

[0062] In the above method, inhibiting, reducing, or silencing the expression of the gene encoding the protein in the plant includes introducing a gene knockout vector with target sequences of 5'-TCTCGCAGCTCGCAGTACCG-3' and 5'-ACTCCCATCTACCAGTCCTG-3' into the target plant.

[0063] In the above text, the gene knockout vector pCBSG015-sgRNA is a recombinant plasmid obtained by inserting DNA fragments with sequences 5'-TCTCGCAGCTCGCAGTACCG-3' and 5'-ACTCCCATCTACCAGTCCTG-3' between the restriction endonuclease BsaI sites of the pCBSG015 vector, while keeping other sequences of the pCBSG015 vector unchanged.

[0064] In one specific embodiment, the method for cultivating soybeans with improved salt tolerance provided by the present invention includes performing a single-vector dual-target double knockout of the genome of the target soybean Dongnong 50 (DN50). The soybean with improved salt tolerance may be a transgenic soybean that has undergone the following mutations:

[0065] 1) Compared with the target soybean, the GmERF416 gene in the genome of the soybean with improved salt tolerance has the following mutation: there is a deletion of one nucleotide at position 513 of SEQ ID No. 1 (corresponding to position 513 of SEQ ID No. 3) on both chromosomes, namely the deletion of nucleotide "G", which causes premature termination of GmERF416 translation, thereby knocking out the GmERF416 gene;

[0066] 2) Compared with the target soybean, the GmERF416 gene in the genome of the soybean with improved salt tolerance has the following mutation: 24 nucleotides are deleted in positions 489-512 of SEQ ID No. 1 (corresponding to positions 489-512 of SEQ ID No. 3) on both chromosomes, namely the deletion of nucleotide "5'-CCGGGGTGTCACCTTCTACCGCAG-3'", which leads to premature termination of GmERF416 translation, thereby knocking out the GmERF416 gene.

[0067] In this invention, the proteins and / or the biological materials described above are also within the scope of protection claimed by this invention.

[0068] In this invention, the plant may be a dicotyledonous plant.

[0069] In the above applications or methods, the dicotyledonous plant may be N1, N2, N3, or N4.

[0070] N1) Leguminosae;

[0071] N2) Leguminosae (family legumes);

[0072] N3) Soybean plants;

[0073] N4) Soybeans.

[0074] The soybeans mentioned above may refer to the soybean variety Dongnong 50.

[0075] This invention constructed a GmERF416 gene knockout vector pCBSG015-sgRNA, which was introduced into Agrobacterium EHA105 competent cells to obtain Agrobacterium EHA105-pCBSG015-sgRNA. Two homozygous plants, erf416-1 and erf416-2, encoding the GmERF416 gene, were obtained by infecting soybean Dongnong 50 (DN50) with Agrobacterium EHA105-pCBSG015-sgRNA. Salt tolerance experiments showed that erf416-1 and erf416-2 exhibited significantly higher salt tolerance than the control Dongnong 50 (DN50). Statistical analysis indicates that GmERF416 negatively regulates soybean salt tolerance, and reducing GmERF416 expression significantly improves soybean salt tolerance. Attached Figure Description

[0076] Figure 1 Transcription of GmERF416 was higher in salt-sensitive soybean materials under 12 hours of salt stress than in salt-tolerant materials.

[0077] Figure 2 Preparation of the GmERF416 gene editing mutant. The specific locations of the two target sequences T1 and T2 (sgRNA1 and sgRNA2) of GmERF416 on the GmERF416 gene and the gene editing verification of the erf416 mutant are shown.

[0078] Figure 3 Molecular detection of homozygous mutants of the GmERF416 gene, erf416-1 and erf416-2.

[0079] Figure 4Phenotypic characteristics of the homozygous mutants of the GmERF416 gene, erf416-1 and erf416-2, during their growth period in saline soil.

[0080] Figure 5 Phenotypic analysis of the GmERF416 gene mutant and control under salt stress during the growth period. A) Leaf area at the first trifoliate leaf stage (V3 stage); B) Plant height at the fifth to sixth trifoliate leaf stage (V5-6 stage); C) Plant height at the sixth to seventh trifoliate leaf stage (V6-7 stage); D) Plant height under salt stress relative to normal conditions at the sixth to seventh trifoliate leaf stage (salt stress condition: 0.5% NaCl). Detailed Implementation

[0081] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0082] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0083] Unless otherwise specified, the quantitative experiments in the following examples are all repeated three times, and the results are averaged.

[0084] The soybean variety Williams 82 (W82) used in the following examples was donated by Professor Scott Jackson of the Department of Agronomy at Purdue University and is described in: Scott A. Jackson et al., Genomesquence of the palaeopolyploid soybean, Nature, 2010, Vol. 463, 178-183. This biological material is available to the public from the applicant and is intended solely for the replication of experiments of this invention and may not be used for any other purpose.

[0085] The soybean variety Dongnong 50 (Glycine max L.Merr.DN50) in the following examples is a new variety bred by Northeast Agricultural University in China. It has been widely recognized and promoted due to its high production performance, stress resistance, and processing quality. It is described in the following literature: Junmei Hu, Yongbin Zhuang, Xianchong Li, Xiaoming Li, Chanchan Sun, Zhaojun Ding, Ran Xu, Dajian Zhang, Time-series

[0086] transcriptome comparison reveals the gene regulation network undersalt

[0087] stress in soybean(Glycine max)roots,BMC Plant Biology,2022Mar

[0088] 31;22(1):157.doi:10.1186 / s12870-022-03541-9。 The biological material is available to the public from the applicant and is intended solely for the purpose of repeating experiments of this invention and may not be used for any other purpose.

[0089] The wild soybean materials Y0532 and Y55 used in the following examples were provided by Researcher Lai Yongcai of the Heilongjiang Academy of Agricultural Sciences and are described in: Bian XH, et al., A class B heat shock factor selected for during soybean domestication contributes to salt tolerance by promoting flavonoid biosynthesis. New Phytologist (2020). 225:268–283. This biological material is available to the public from the applicant and is intended solely for the purpose of replicating experiments of this invention; it may not be used for any other purpose.

[0090] The *Agrobacterium tumefaciens* EHA105 in the following examples is described in the following literature: Rodrigues SD, Karimi M, Imppens L, Van Lerberge E, Coussens G, Aesaert S, Rombaut D, Holtappels D, Ibrahim HMM, Van Montagu M, Wagemans J, Jacobs TB, De Coninck B, Pauwels L., *Efficient CRISPR-mediated base editing in *Agrobacterium spp.*, Proc Natl Acad Sci USA. 2021 Jan, 12; 118(2):e2013338118. doi:10.1073 / pnas.2013338118. This biological material is available to the public from the applicant and is intended solely for the purpose of repeating experiments of this invention and shall not be used for any other purpose.

[0091] The pCBSG015 vector used in the following examples was provided by Wimi Biotechnology Co., Ltd., catalog number wimi-pCXB053.

[0092] The following examples used SPSS 11.5 statistical software to process the data. The experimental results are expressed as mean ± standard deviation. One-way ANOVA was used. P < 0.05 (*) indicates a significant difference, and P < 0.01 (**) indicates a highly significant difference.

[0093] Example 1: Obtaining the soybean GmREF416 gene

[0094] 1. Cloning of the soybean GmREF416 gene

[0095] Primers were designed based on the Williams 82 (W82) reference genome sequence (Genome Assebly Glycein__max_v4.0_Genbank: GCA_000004515.5, updated: March 10, 2021).

[0096] ERF416-CDS-F: 5'-ATGGCTATGTTTGATCTCAATGTT-3';

[0097] ERF416-CDS-R: 5'-TTAATATGTGTGCTGATTGGAAGTG-3'.

[0098] The GmERF416 gene was amplified from total soybean RNA using ERF416-CDS-F / ERF416-CDS-R primers: W82 leaves were crushed in liquid nitrogen, suspended in 4 mol / L guanidine thiocyanate, and extracted with acidic phenol and chloroform. Anhydrous ethanol was added to the supernatant for precipitation, and the precipitate was dissolved in water to obtain total RNA. 1 μg of total RNA was reverse transcribed using a Thermofisher reagent kit according to the kit instructions. The resulting cDNA fragment was used as a template for PCR amplification.

[0099] The 50 μl PCR reaction system consists of: 1 μl cDNA (0.05 μg), 1.5 μl of the above primers (10 μM), 25 μl 2× PCR buffer, 10 μl dNTP (10 mM) and 1 U KOD DNA polymerase, and then made up to 50 μl with ultrapure water.

[0100] The reaction was performed on a PE9600 PCR instrument with the following program: denaturation at 94℃ for 5 min; followed by 1 min at 98℃, 1 min at 58℃, and 1 min at 68℃, for a total of 30-32 cycles; then extension at 68℃ for 10 min; and storage at 4℃.

[0101] A PCR product of approximately 1200 bp was obtained. Sequence analysis of the recovered PCR product revealed it to be 1251 bp, possessing the nucleotide sequence of SEQ ID No. 1, which is the encoding gene for GmERF416. The encoded protein is named GmERF416. The amino acid sequence of GmERF416 is SEQ ID No. 2. The genomic sequence of the GmERF416 gene is SEQ ID No. 3. Positions 1-539, 1689-1714, 1856-1886, 1993-2080, 2164-2312, 3033-3136, 3278-3408, and 4421-4603 in SEQ ID No. 3 are exons; the remaining nucleotides are introns.

[0102] 2. Transcription of the GmREF416 gene in salt-sensitive and salt-tolerant soybeans under salt stress

[0103] Salt-sensitive wild soybean Y0532 and salt-tolerant wild soybean Y55 were sown in vermiculite. Two weeks after emergence, the vermiculite was washed off the roots, and the seedlings were hydroponically cultured for two days to recover growth. Then, the seedlings were treated with 150 mM NaCl for 3, 6, and 12 hours, respectively. RNA was extracted from the roots at the time points after salt treatment for transcriptome sequencing.

[0104] FPKM (Fragments Per Kilobase of exon per Million fragments mapped) is a widely used unit for reporting gene expression levels in the sequencing field. It measures the expression level of a gene (or transcript) by dividing the amplification of sequencing fragments on a chromosome into a numerical index. FPKM is a standardized unit of measurement; the FPKM ratio can measure the expression level of a gene under specific conditions. FPKM can also be used to compare the expression levels of different genes or between genes, as well as the differences in expression levels between different biological samples. Therefore, FPKM is a commonly used method for representing gene expression results and can reflect the gene expression status. Its calculation method is: reads per million mapped per thousand bases of transcription. FPKM values ​​can reflect the expression level of a specific gene under specific experimental conditions and have relatively good reliability.

[0105] The FPKM of GmERF416 was calculated based on the above transcriptome sequencing results, and the results are shown in [the table below]. Figure 1 Under normal conditions, the FPKM values ​​of Y0532 and Y55 are approximately 2.6 and 3.9, respectively. The transcription level of GmERF416 in the salt-tolerant material Y55 is significantly higher than that in the salt-sensitive material Y0532. After 3 hours of treatment with 150 mM NaCl, the transcription levels of both materials significantly increased, reaching approximately 4.9 and 5.2, respectively, while the FPKM values ​​of the two materials were similar. After 6 hours of salt treatment, the transcription level of GmERF416 in both materials continued to rise, reaching approximately 8.5. After 12 hours of salt treatment, the FPKM value in Y0532 rose sharply to 12.5, while it decreased to 6.4 in Y55. These results indicate that after 12 hours of salt stress, the transcription level of GmERF416 in the salt-tolerant material Y55 was significantly less induced than that in the salt-sensitive material Y0532. GmERF416 and its encoding gene may negatively regulate plant salt tolerance.

[0106] Example 2: Obtaining the GmERF416 gene mutant using gene editing technology

[0107] 1. Construction of the pCBSG015 gene editing vector

[0108] Target sequence selection: The high-throughput CRISPR-Cas9 target design program developed by Vimi was used. The target design principles of this program are as follows: 1) The knockout site is located in the coding sequence (CDS) region and preferably at the protein front or in an important functional domain; 2) It should cover a higher proportion of transcripts; 3) There should be no off-target effects or the off-target effects should be located in intergenic regions; 4) Targets with higher editing efficiency should be preferred; 5) The sequence should have a relatively balanced GC content and be less prone to secondary structure formation.

[0109] A dual-gene, dual-target knockout approach was adopted. The selected target sites were both located in the first exon region of the GmERF416 gene, with target sequences T1 and T2 (nucleotides 472-494 and 507-529 of SEQ ID No. 1, which are also nucleotides 472-494 and 507-529 of SEQ ID No. 3). The positions of T1 and T2 in SEQ ID No. 1 are shown below. Figure 2 As shown.

[0110] T1:5'-TCTCGCAGCTCGCAGTACCG-3';

[0111] T2:5'-ACTCCCATCTACCAGTCCTG-3'.

[0112] Promoter selection: AtU6 derived from Arabidopsis thaliana was used to promote the T1 and T2 target sites.

[0113] Preparation of sgRNA expression cassettes containing target sites: pCBSG015 vector was linearized by Bsa I restriction enzyme digestion. The T1 sequence was directly synthesized using primer synthesis method, and 16bp vector sequences were added to both ends as homologous arms (U6-T1, U6-T2). Reverse complementary sequences (Anti-U6-T1, Anti-U6-T2) were synthesized, annealed to form double strands, and homologously recombinated with the backbone linear vector.

[0114] The specific steps are as follows:

[0115] Sense-U6-T1: 5'-ggcaccgagtcggtgcTCTCGCAGCTCGCAGTACCGgttgaacaacggaaac-3';

[0116] Anti-U6-T1: 5'-gtttccgttgttcaacCGGTACTGCGAGCTGCGAGAgcaccgactcggtgcc-3';

[0117] Lowercase letters represent homologous arms, and uppercase letters represent T1 sequences.

[0118] Sense-U6-T2: 5'-ggcaccgagtcggtgcACTCCCATCTACCAGTCCTGgttgaacaacggaaac-3';

[0119] Anti-U6-T2: 5'-gtttccgttgttcaacCAGGACTGGTAGATGGGAGTgcaccgactcggtgcc-3';

[0120] Lowercase letters represent homologous arms, and uppercase letters represent T2 sequences.

[0121] Preparation of annealed AtU6-T1-gRNA and AtU6-T2-gRNA fragments: The synthesized Sense-U6-T1 and Anti-U6-T1, Sense-U6-T2 and Anti-U6-T2 sequences were annealed to form double strands. The reaction system was as follows: the synthesized sequences were dissolved in 75 mM NaCl solution to a final concentration of 0.2 nM / μl, and equal volumes of the forward and reverse strand solutions (Sense-U6-T1 and Anti-U6-T1, Sense-U6-T2 and Anti-U6-T2) were mixed. The mixture was heated in a 95℃ water bath for 5-10 min, followed by slow cooling to obtain the annealed AtU6-T1-gRNA and AtU6-T2-gRNA fragments.

[0122] Vector linearization by restriction enzyme digestion: 1-2 μg of pCBSG015 plasmid, 10×CutSmart TM 5 μl of buffer (NEB), 1 μl of Bsa I restriction enzyme, and sterile double-distilled water were added to make up to 50 μl. The mixture was reacted in a water bath at 37°C for 30 min, and then purified using the EZ-10 Column DNA Purification Kit (Shanghai Sangon Biotech). The purified DNA was dissolved in an appropriate amount of water to obtain the linearized pCBSG015 vector.

[0123] Homologous recombination of the target sgRNA expression cassette with the pCBSG015 vector was performed using the EasyGeno Rapid Recombinant Cloning Kit (TianGen). The reaction mixture consisted of 5 μL of 2×EasyGeno Assembly mix buffer, 0.5 μL of linearized pCBSG015 vector, and 4.5 μL of annealed AtU6-T1-gRNA and AtU6-T2-gRNA. The reaction conditions were 50 °C for 15 min. The ligation product was transformed into E. coli DH5α competent cells, and plasmids were extracted from positive colonies. After successful sequencing, the recombinant vector pCBSG015-sgRNA was obtained.

[0124] The recombinant vector pCBSG015-sgRNA is a recombinant plasmid obtained by replacing the fragment between 5'-ggcaccgagtcggtgc-3' and 5'-gttgaacaacggaaac-3' of the pCBSG015 vector with the DNA sequence of SEQ ID No. 4, while keeping the other sequences of the pCBSG015 vector unchanged. The recombinant plasmid is named recombinant vector pCBSG015-sgRNA.

[0125] 2. Genetic transformation of soybeans

[0126] The recombinant vector pCBSG015-sgRNA was transformed into Agrobacterium EHA105 competent cells to obtain Agrobacterium EHA105-pCBSG015-sgRNA.

[0127] Soybean variety DN50 was infected with the Agrobacterium EHA105-pCBSG015-sgRNA prepared above to obtain soybean plants with the GmERF416 gene edited, a process performed by Baige Company. T0 generation seeds were harvested, and T1 generation seeds were obtained through self-pollination. T1 generation seedlings were then planted to obtain T2 generation seedlings, which were then subjected to the following tests. Although DN50 is a Northeast China variety and not suitable for growth in Beijing greenhouses, it is easily transformed, has a short growth cycle, and exhibits some salt tolerance; therefore, it was used as a recipient control.

[0128] 3. Screening of homozygous GmERF416 gene mutations

[0129] Using the genomic DNA of the T2 generation GmERF416 gene-edited seedlings obtained in step 2 as a template, PCR primers designed approximately 219 bp upstream and 690 bp downstream of the target sequence T1 were used to amplify the DNA sequence of approximately 932 bp near the target sequence and sequenced to detect the GmERF416 gene editing method. The PCR identification primer set for the target site is as follows: Cas-F: 5'-CAAAACATGGCACCACAAGAG-3', Cas-R: 5'-GAGAAAACTGAATAAAAAGGTTCATTTT-3'.

[0130] After successful sequencing, the CRISPR target editing method was analyzed using the website DSDecode (http: / / dsdecode.scgene.com / ) and compared with the standard gene sequence using manual peak reading. The editing methods of each target sequence and its upstream and downstream sequences were analyzed and passaged.

[0131] The gene editing methods for the GmERF416 gene homozygous mutants erf416-1 and erf416-2, screened through the above method, are as follows: Figure 1 As shown.

[0132] Compared with the recipient soybean variety DN50, the GmERF416 gene in the erf416-1 mutant genome has a mutation: in two homologous chromosomes, there is a deletion of one nucleotide at position 513 of SEQ ID No.1 (corresponding to position 513 of SEQ ID No.3), namely the deletion of nucleotide "G", which causes premature termination of GmERF416 translation, thereby knocking out the GmERF416 gene.

[0133] Compared with the recipient soybean variety DN50, the GmERF416 gene in the erf416-2 mutant genome has a mutation: a 24-nucleotide deletion exists in positions 489-512 of SEQ ID No.1 (corresponding to positions 489-512 of SEQ ID No.3) on both homologous chromosomes, namely the deletion of the nucleotide “5'-CCGGGGTGTCACCTTCTACCGCAG-3'”, which causes premature termination of GmERF416 translation, thereby knocking out the GmERF416 gene.

[0134] Seeds from T2 generation plants (T3 generation) of mutants erf416-1 and erf416-2 were harvested for subsequent experiments.

[0135] Example 3: Phenotypic analysis of CRISPR mutants erf416-1 and erf416-2 of the GmERF416 gene

[0136] 1. Detection of GmERF416 gene expression levels in erf416-1 and erf416-2

[0137] Total RNA was extracted from mid-development seeds of soybean varieties DN50 and erf416-1 and erf416-2 and reverse transcribed. The cDNA obtained from the reverse transcription was used as a template, and the primers were: ERF416-qF: 5'-AGAGGTGCATTAGCCCTTCG-3'; ERF416-qR: 5'-CCAACATGACAGGCACTTGC-3'. Real-time PCR was used to identify the expression level of the GmERF416 gene.

[0138] The soybean Tublin gene was used as an internal control. The internal control primers were Primer-TF: 5'-TGGCCGTTACCTGACAGCAT-3' and Primer-TR: 5'-CTCGGAGGGATGTCACACAC-3'. The results are as follows: Figure 3 As shown, the relative expression level of GmERF416 in DN50 was 0.015, while the expression levels in erf416-1 and erf416-2 were approximately 0.0043 and 0.0052, respectively. This indicates that the expression level of GmERF416 in erf416-1 and erf416-2 was significantly decreased. Figure 3 ).

[0139] 2. Effects of the GmERF416 gene on the salt tolerance of soybean

[0140] Salt tolerance was tested in a greenhouse. The first-generation seeds of erf416-1 and erf416-2 and the control soybean variety DN50 were planted in the greenhouse. The control group was irrigated with water, while the soil salinity (excluding the original soil salinity) of the experimental group was adjusted to 0.5% with NaCl.

[0141] The specific procedure was as follows: The soil was dried thoroughly and weighed to 35 kg. 0.175 kg of NaCl (0.5%) was dissolved in 35 kg of water, and the solution was poured into the soil, mixing it evenly. The control and mutant plants were directly sown in the same pot with soil, with four plants per pot (four replicates). The soil moisture content was maintained at 70-75% during the growth process. Greenhouse conditions: light duration 16h:8h (daytime:nighttime); temperature: 30-37℃ during the day and 25-28℃ at night. The growth status at each stage was observed. The biological experiment was repeated three times, and the results were expressed as mean ± standard deviation. A one-way ANOVA test was used; P < 0.05 (*) indicated a significant difference, and P < 0.01 (**) indicated a highly significant difference.

[0142] Figure 4 The image shows the growth status of the homozygous mutants erf416-1 and erf416-2 of the control DN50 and GmERF416 genes at the first trifoliate leaf stage (V3), the fifth-sixth trifoliate leaf stage (V5-6), and the sixth-seventh trifoliate leaf stage (V6-7) in soil containing 0.5% NaCl. This indicates that the growth of mutants erf416-1 and erf416-2 is significantly better than that of the control in soil containing 0.5% NaCl.

[0143] The phenotypic analysis results of the homozygous mutants erf416-1 and erf416-2 of the DN50 and GmERF416 genes at different growth stages in soil containing 0.5% NaCl are as follows:

[0144] 1) Under normal conditions, the leaf areas of DN50 at the first trifoliate compound leaf stage (V3 stage) and mutants erf416-1 and erf416-2 were 9.8±4.9, 10.5±2.3, and 9.0±2.4 cm², respectively. 2 In soil containing 0.5% NaCl, the leaf areas of DN50 and the mutants erf416-1 and erf416-2 were 5.7±2.5, 9.0±2.6, and 9.0±3.6 cm², respectively. 2 Compared to the control, DN50 and its mutants erf416-1 and erf416-2 showed a decrease in leaf area of ​​approximately 42%, 14%, and 0%, respectively, under salt stress. The salt tolerance of erf416-1 and erf416-2 was significantly higher than that of the control GN50. Figure 5 (A)

[0145] 2) Under normal conditions, the plant heights of DN50 and mutants erf416-1 and erf416-2 at the 5th-6th trifoliate leaf stage (V5-6 stage) were 26.0±7.5 cm, 31.9±9.8 cm, and 26.6±7.5 cm, respectively. In soil containing 0.5% NaCl, the plant heights of DN50 and mutants erf416-1 and erf416-2 were 14.1±3.5 cm, 24.1±12.6 cm, and 22.3±7.4 cm, respectively. Under salt stress, the plant heights of DN50 and mutants erf416-1 and erf416-2 decreased by approximately 46%, 24%, and 19%, respectively, compared to the control. The salt tolerance of erf416-1 and erf416-2 was significantly higher than that of the control GN50. Figure 5 (B)

[0146] 3) Under normal conditions, the plant heights of DN50 and mutants erf416-1 and erf416-2 at the 6th-7th trifoliate leaf stage (V6-7 stage) were 34.9±9.2, 40.7±13.8, and 37.9±6.5 cm, respectively. In soil containing 0.5% NaCl, the plant heights of DN50 and mutants erf416-1 and erf416-2 were 21.5±4.1, 32.5±13.5, and 32.2±9.4 cm, respectively. Compared with normal conditions, the relative plant heights of DN50 and mutants erf416-1 and erf416-2 in saline soil were approximately 62%, 80%, and 82%, respectively, representing decreases of 38%, 20%, and 17%. The salt tolerance of erf416-1 and erf416-2 was significantly higher than that of the control DN50. Figure 5 (C and D in the middle).

[0147] The above statistics show that the number of pods per plant in erf416-1 and erf416-2 was significantly higher than that in the recipient DN50. The yield per plant in erf416-1 and erf416-2 was also significantly higher than that in the control DN50. This indicates that GmERF416 negatively regulates soybean salt tolerance, and reducing the expression level of the gene encoding GmERF416 can significantly improve soybean salt tolerance.

[0148] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. The use of a protein or a substance that regulates gene expression, or a substance that regulates the activity or content of said protein, in any of the following: 1) Application in regulating plant salt tolerance; 2) Application in the preparation of products that regulate plant salt tolerance; 3) Application in cultivating plants with altered salt tolerance; 4) Application in the preparation of products using plants with altered salt tolerance; 5) Applications in plant breeding; The protein is any of the following proteins: a1) A protein with the amino acid sequence SEQ ID No. 2; a2) A protein having the same function as the amino acid sequence shown in SEQ ID No. 2, by substitution and / or deletion and / or addition of one or more amino acid residues; a3) is a protein that has more than 80% identity with the amino acid sequence defined by a1) or a2) and has the same function; a4) A fusion protein obtained by attaching a tag to the end of any of the proteins defined in a1)-a3).

2. The application according to claim 1, characterized in that: The protein is derived from soybeans.

3. The application according to claim 1 or 2, characterized in that: The substance that regulates gene expression or the substance that regulates the activity or content of the protein is a biological material related to the protein in the application of claim 1 or 2, and the biological material is any one of the following: c1) The nucleic acid molecule encoding the protein; c2) An expression cassette containing the nucleic acid molecule described in c1); c3) A recombinant vector containing the nucleic acid molecule described in c1), or a recombinant vector containing the expression cassette described in c2); c4) Recombinant microorganisms containing the nucleic acid molecules described in c1), or recombinant microorganisms containing the expression cassette described in c2), or recombinant microorganisms containing the recombinant vector described in c3); c5) A transgenic plant cell line containing the nucleic acid molecule described in c1), or a transgenic plant cell line containing the expression cassette described in c2); c6) Transgenic plant tissue containing the nucleic acid molecules described in c1), or transgenic plant tissue containing the expression cassette described in c2); c7) A transgenic plant organ containing the nucleic acid molecule described in c1), or a transgenic plant organ containing the expression cassette described in c2); e1) Nucleic acid molecules that inhibit, reduce, or silence the expression of the protein-encoding gene; e2) An expression cassette containing the nucleic acid molecule described in e1); e3) A recombinant vector containing the nucleic acid molecule described in e1), or a recombinant vector containing the expression cassette described in e2); e4) Recombinant microorganisms containing the nucleic acid molecules described in e1), or recombinant microorganisms containing the expression cassette described in e2), or recombinant microorganisms containing the recombinant vector described in e3); e5) A transgenic plant cell line containing the nucleic acid molecule described in e1), or a transgenic plant cell line containing the expression cassette described in e2); e6) Transgenic plant tissue containing the nucleic acid molecules described in e1), or transgenic plant tissue containing the expression cassette described in e2); e7) A transgenic plant organ containing the nucleic acid molecule described in e1) or a transgenic plant organ containing the expression cassette described in e2).

4. The application according to claim 3, characterized in that: c1) The nucleic acid molecule is any of the following DNA molecules. d1) The nucleotide sequence is the DNA molecule shown in SEQ ID No. 3; d2) The coding region sequence is the DNA molecule shown in SEQ ID No. 1; d3) has 90% or more identity with the nucleotide sequence defined by d1) or d2) and is a DNA molecule encoding the protein of claim 1; d4) Hybridizes under stringent conditions to a nucleotide sequence defined by d1) or d2) and encodes a DNA molecule that encodes the protein of claim 1.

5. A method for improving the salt tolerance of plants, characterized in that: The method includes step M, which is to inhibit or reduce or silence the activity and / or content of the protein described in claim 1 or 2 in the target plant, or / and, inhibit or reduce or silence the expression level of the gene encoding the protein described in claim 1 or 2, in order to improve the salt tolerance of the plant.

6. A method for reducing the salt tolerance of plants, characterized in that: The method includes step P, which is to enhance, increase or upregulate the activity and / or content of the protein described in claim 1 or 2 in the target plant, or / and enhance, increase or upregulate the expression level of the gene encoding the protein described in claim 1 or 2, so as to reduce the salt tolerance of the plant.

7. A breeding method for cultivating plants with improved salt tolerance, characterized in that: This includes inhibiting, reducing, or silencing the expression level of the gene encoding the protein of claim 1 or 2 in the target plant, and / or, the activity and / or content of the protein resulting in a plant with enhanced salt tolerance, wherein the salt tolerance of the enhanced plant is higher than that of the recipient plant.

8. The method according to claim 7, characterized in that: Includes the following steps: (1) Construct a recombinant expression vector for the gene encoding the protein described in claim 1 or 2 that inhibits, reduces or silences it; (2) The recombinant expression vector constructed in step (1) is transferred into the recipient plant to obtain a plant with higher salt tolerance than the recipient plant.

9. The protein as described in claim 1 or 2 and / or the biomaterial as described in claim 3 or 4.

10. The application according to any one of claims 1-4, and / or the method according to any one of claims 5-8, characterized in that: The plant is any one of the following: N1) Dicotyledons N2) Leguminosae; N3) Leguminosae (family legumes); N4) Soybean genus plants; N5) soybeans.

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