Application of protein GmHSF35 and related biological materials in regulating protein content in plant seeds

Abstract

The present application relates to the field of variation or genetic engineering, and discloses a protein GmHSF35 with an amino acid sequence of SEQ ID NO: 2 and related biological materials of the protein in regulating plant seed protein accumulation and cultivating plants with increased seed protein content. Experiments prove that the protein content in plant seeds can be increased by knocking out the GmHSF35 gene, and the GmHSF35 protein negatively regulates the protein content in plant seeds, which has important significance for cultivating high-protein plant varieties, especially high-protein plant varieties in plant seeds.

Description

Technical Field

[0001] This invention relates to the field of mutation or genetic engineering, and in particular to the application of protein GmHSF35 and related biomaterials in regulating protein content in plant seeds. Background Technology

[0002] Soybeans are rich in nutrients and are an important economic crop providing edible oil and plant protein. Soybean protein is a plant-based protein with an amino acid composition similar to milk protein. Except for a slightly lower level of methionine, it is rich in all other essential amino acids, contains no cholesterol, and contains various physiologically active substances such as isoflavones, soy saponins, soy phospholipids, and phytosterols. These active substances play various physiological regulatory roles in the human body, helping to boost immunity, protect cardiovascular health, and promote digestion.

[0003] Soybean protein has a genetic structure that closely resembles the amino acids in the human body, making it one of the most nutritious plant proteins. It is widely used in various food systems, including soy milk and other soy products, and its emulsifying properties make it suitable as an additive in meat products, baked goods, dairy products, and beverages. Therefore, increasing the protein content of soybean seeds is one of the main directions in soybean breeding.

[0004] Ma et al. obtained 58 HSF sequences from the soybean genome. After removing sequences lacking typical HSF DBD domains and coiled-coil domains that mediate protein interactions, they identified 38 heat shock transcription factors, named GmHSF1-38. Glyma.17G174900 is annotated in NCBI as NM_001353466.1, GmHSF35 (Pytozome annotation: Heat stress transcription factor B-1, heat shock transcription factor). GmHSF34 is known to regulate heat and drought tolerance in plants. Overexpression of GmHSF34 in Arabidopsis thaliana enhances the heat and drought tolerance of transgenic plants (Li et al. BMCGenomics 2014, 15:1009). GmHSFB2b regulates the accumulation of flavonoids and stress tolerance in plants. No reports have been found regarding the regulation of protein content in seeds by these genes. Summary of the Invention

[0005] The technical problem to be solved by this invention is how to increase the protein content in plant seeds.

[0006] To address the aforementioned problems, the present invention provides the application of a protein, a substance regulating the expression of a gene encoding the protein, or a substance regulating the activity or content of the protein, wherein the application may be any of the following:

[0007] A1) Application in regulating protein content in plant seeds;

[0008] A2) Application in the preparation of products that regulate the protein content of plant seeds;

[0009] A3) Application in cultivating plants with altered protein content in seeds;

[0010] A4) Application in the preparation of products from plants whose protein content has been altered in the seeds;

[0011] A5) Applications in plant breeding;

[0012] The protein is GmHSF35, and can be any of the following proteins:

[0013] B1) A protein with the amino acid sequence SEQ ID NO: 2;

[0014] B2) A protein with the same function as the amino acid sequence shown in SEQ ID NO: 2, but with amino acid residue substitution and / or deletion and / or addition.

[0015] B3) Proteins that share more than 80% identity with the amino acid sequence defined by B1) or B2) and have the same function;

[0016] B4) A fusion protein obtained by attaching a tag to the end of any of the proteins defined in B1)-B3).

[0017] The indicators for plant breeding include the protein content in plant seeds, and the purpose of plant breeding includes cultivating plants with altered protein content in their seeds.

[0018] 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.

[0019] Of the proteins mentioned above, sequence 2 (SEQ ID NO: 2, 282aa) is named GmHSF35 protein or protein GmSHF35, specifically as follows: MSQQQRSTPAPFLTKTYLLVDDPATDDVVSWSEGGNTFVVWKHADFAKDLLPKYFKHNNFSSFVRQLNTY GFRKTVPDKWEFANEYFKRGQTDLLAEIRRRKVVSPVTGKSTGGGVNISASHSGGDDMGSTSTGSMEAATAAGADISGEN EKLKKDNEKLSGELALAKKQCEELVAFLRDSLNVSPDVIERIIRQGTCVSSNNDAVRFDAIINNDLDDDENAVGECEPQQ PPPKGDNGESLKLFGVWLKGQNGKENSIGKCRKRGRQDYPIAATAKELKTLV.

[0020] In the above applications, the protein may be derived from soybean (Glycine max(L.)Merr.).

[0021] In the above text, the substance that regulates the expression of the gene encoding the protein or the substance that regulates the activity or content of the protein may be a substance that performs at least one of the following six types of regulation: C1) regulation at the transcriptional level of the gene; C2) post-transcriptional regulation of the gene (i.e., regulation of splicing or processing of the primary transcript of the gene); C3) regulation of RNA transport of the gene (i.e., regulation of mRNA transport of the gene from the nucleus to the cytoplasm); C4) regulation of translation of the gene; C5) regulation of mRNA degradation of the gene; C6) post-translational regulation of the gene (i.e., regulation of the activity of the protein translated from the gene).

[0022] In the above applications, the substance regulating the expression of the protein-coding gene can be any of the following:

[0023] D1) Nucleic acid molecules that inhibit, reduce, or silence the expression of the protein-encoding genes described above;

[0024] D2) An expression cassette containing the nucleic acid molecules described in D1);

[0025] D3) A recombinant vector containing the nucleic acid molecule described in D1), or a recombinant vector containing the expression cassette described in D2);

[0026] D4) Recombinant microorganisms containing the nucleic acid molecules described in D1), or recombinant microorganisms containing the expression cassette described in D2), or recombinant microorganisms containing the recombinant vector described in D3);

[0027] D5) A transgenic plant cell line containing the nucleic acid molecule described in D1), or a transgenic plant cell line containing the expression cassette described in D2);

[0028] D6) Transgenic plant tissue containing the nucleic acid molecules described in D1), or transgenic plant tissue containing the expression cassette described in D2);

[0029] D7) Transgenic plant organs containing the nucleic acid molecules described in D1) or transgenic plant organs containing the expression cassette described in D2).

[0030] Those skilled in the art can readily mutate the nucleotide sequence of the present invention that inhibits, reduces, or downregulates the expression of the GmHSF35 protein encoding gene using known methods, such as directed evolution or point mutation. Nucleotides that are artificially modified and possess 80% or more of the identity with the nucleotide sequence of the present invention that inhibits, reduces, or downregulates the expression of the GmHSF35 protein encoding gene, and which have the function of inhibiting, reducing, or downregulating the expression of the GmHSF35 protein encoding gene, are all derived from and equivalent to the nucleotide sequence of the present invention.

[0031] 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.

[0032] 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.

[0033] 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.

[0034] In the above applications, the nucleic acid molecule described in D1) can be any of the following DNA molecules:

[0035] F1) The nucleotide sequence is the DNA molecule shown in SEQ ID NO: 3;

[0036] F2) The coding region sequence is the DNA molecule shown in SEQ ID NO: 1 in the sequence listing;

[0037] F3) has 90% or more identity with the nucleotide sequence defined by F1) or F2) and is a DNA molecule encoding the protein described above;

[0038] F4) hybridizes under strict conditions with a nucleotide sequence defined by F1) or F2) and is a DNA molecule encoding the protein described above.

[0039] In the above applications, the nucleic acid molecule described in D1) is a gRNA that targets the protein-coding gene described above, and the target sequence of the gRNA may be 5′-TCCTCGCTTGAAATACTCATTGG-3′ (sequence 3, positions 2953-2975) or / and 5′-GTGACCGGAAAATCTACCGGCGG-3′ (sequence 3, positions 3024-3046).

[0040] 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.

[0041] In D3) above, the recombinant vector can be a plant gene editing vector. The plant gene editing vector can be the knockout vector WMC024-sgRNA. In this invention, the WMC024-sgRNA vector contains the AtU6 promoter and carries the diammonium phosphate resistance gene.

[0042] In this invention, the knockout is performed using a single-gene dual-target knockout method.

[0043] The recombinant vector WMC024-sgRNA is a recombinant plasmid obtained by inserting DNA fragments containing the sequences 5′-TCCTCGCTTGAAATACTCATTGG-3′(T1) and 5′-GTGACCGGAAAATCTACCGGCGG-3′(T2) between the restriction endonuclease Bsa I sites of the WMC024 vector, while keeping other sequences of the WMC024-sgRNA vector unchanged.

[0044] The target sequence of T1 is the DNA fragment at positions 2953-2975 of sequence 3, and the target sequence of T2 is the DNA fragment at positions 3024-3046 of sequence 3.

[0045] The microorganism mentioned in D4) above can be Agrobacterium. The Agrobacterium is EHA105.

[0046] The present invention also provides a method for increasing the protein content in plant seeds.

[0047] The present invention provides a method for regulating the protein content in plant seeds, the method comprising step M, wherein step M is to inhibit or reduce or silence the activity and / or content of the protein in the target plant, or / and, inhibit or reduce or silence the expression level of the gene encoding the protein, thereby increasing the protein content in plant seeds.

[0048] Suppressing, reducing, or downregulating the expression of the coding gene can be achieved through gene knockout or gene silencing.

[0049] 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) refers to a cluster of regularly spaced short palindromic repeat sequences, which is a site in the genome containing multiple short repeat sequences. The Cas9 protein, mediated by RNA, can cleave target sequences recognized by crRNA–tracrRNA.

[0050] The present invention also provides a breeding method for cultivating plants with increased protein content in their seeds. The method includes step M, wherein step M is to inhibit or reduce or silence the activity and / or content of the protein in the target plant, or / and, inhibit or reduce or silence the expression level of the gene encoding the protein, in order to increase the protein content in the plant seeds. The plant with increased protein content in its seeds has a higher protein content in its seeds than the target plant.

[0051] In the above method, step M includes introducing a gene knockout vector into the target plant with nucleotides 2953-2975 and / or nucleotides 3024-3046 of sequence 3 in the sequence listing as the target sequence.

[0052] In the above method, the genome of the target plant contains a DNA molecule whose nucleotide sequence is sequence 3 in the sequence listing, and step M includes performing any of the following operations on the genome of the target plant:

[0053] E1) Delete the four deoxyribonucleotides GTAT between positions 2960-2963 of sequence 3 in the sequence listing of the target plant genome;

[0054] E2) Delete 80 deoxyribonucleotides between positions 2960-3093 of sequence 3 in the target plant genome sequence listing: GTATTTCAAGCGAGGACAGACGGATCTTCTCGCCGAGATCAGGCGGCGGAAGGTGGTGTCTCCGGTGACCGGAAAATCTA.

[0055] In one embodiment of the present invention, the breeding method for cultivating plants with increased protein content in their seeds includes the following steps:

[0056] (1) Construct recombinant expression vectors that suppress, reduce, or silence the proteins described above;

[0057] (2) The recombinant expression vector constructed in step (1) is transferred into the recipient plant to obtain a plant with a higher protein content in the seeds than the recipient plant.

[0058] In the above text, the gene knockout vector WMC024-sgRNA is a recombinant plasmid obtained by inserting DNA fragments with sequences 5′-TCCTCGCTTGAAATACTCATTGG-3′ and 5′-GTGACCGGAAAATCTACCGGCGG-3′ between the restriction endonuclease Bsa I restriction sites of the WMC024 vector, while keeping other sequences of the WMC024 vector unchanged.

[0059] In one specific embodiment, the method for cultivating soybeans with increased protein content in seeds provided by the present invention includes performing a single-vector dual-target double knockout of the genome of the target soybean material Jack. The soybeans with increased protein content in seeds can be transgenic soybeans that have undergone the following mutations:

[0060] 1) Compared with the target soybean, the GmHSF35 gene in the genome of the soybean with increased protein content in the seed has the following mutation: 4 deoxyribonucleotides GTAT are deleted between positions 2960-2963 of SEQ ID NO: 3 in two chromosomes, which leads to premature termination of GmHSF35 translation, thereby knocking out the GmHSF35 gene.

[0061] 2) Compared with the target soybean, the GmHSF35 gene in the genome of the soybean with increased protein content in the seed has the following mutation: 80 nucleotides are deleted at positions 2960-3093 of SEQ ID NO: 3 on both chromosomes, namely the deletion of nucleotide “5′-GTATTTCAAGCGAGGACAGACGGATCTTCTCGCCGAGATCAGGCGGCGGAAGGTGGTGTCTCCGGTGACCG GAAAATCTA-3′”, which causes premature termination of GmHSF35 translation, thereby knocking out the GmHSF35 gene.

[0062] In this invention, the proteins and / or the biological materials described above also fall within the scope of protection claimed by this invention.

[0063] In the above applications or methods, the dicotyledonous plant may be N1) or N2) or N3) or N4) or N5): N1) dicotyledonous plant; N2) legume; N3) legume; N4) soybean; N5) soybean.

[0064] This invention constructed a GmHSF35 gene knockout vector, WMC024-sgRNA, and introduced it into Agrobacterium EHA105 competent cells to obtain Agrobacterium EHA105-WMC024-sgRNA. Soybean Jack was infected with Agrobacterium EHA105-WMC024-sgRNA to obtain two homozygous plants, hsf35-1 and hsf35-2, with the GmHSF35-encoding gene knocked out. Experiments showed that compared with the control Jack, the protein content in the seeds of hsf35-1 and hsf35-2 was significantly higher. Statistical analysis indicates that GmHSF35 negatively regulates the protein content in soybean seeds, and reducing the expression level of GmHSF35 can significantly increase the protein content in soybean seeds. Attached Figure Description

[0065] Figure 1 Preparation of the GmHSF35 gene-editing mutant. The specific locations of the two target sequences T1 and T2 of GmHSF35 on the GmHSF35 gene are shown, along with gene editing verification of the hsf35 mutant.

[0066] Figure 2 To detect the expression level of GmSHF35 in the homozygous mutants hsf35-1 and hsf35-2 of the GmHSF35 gene.

[0067] Figure 3 Protein content was determined in seeds of the homozygous mutants hsf35-1 and hsf35-2 of the GmHSF35 gene. Detailed Implementation

[0068] 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.

[0069] 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.

[0070] Unless otherwise specified, all percentages in the following examples refer to mass percentages in quantitative tests.

[0071] In the quantitative experiments in the following examples, three replicates were set up, and the data are the mean or mean ± standard deviation of the three biological replicates.

[0072] The soybean variety Jack (Glycine max L.Merr.Jack) used in the following examples is described in the following literature: Jian Han, Bingfu Guo, Yong Guo, Bo Zhang, Xiaobo Wang, and Li-Juan Qiu, Creation of Early Flowering Germplasm of Soybean by CRISPR / Cas9 Technology, Front Plant Sci. 2019; 10:1446 Published online 2019Nov22. doi:10.3389 / fpls.2019.01446.

[0073] The soybean variety HN44 in the following examples was obtained from the Soybean Research Institute of Heilongjiang Academy of Agricultural Sciences. It is a soybean variety approved by the Heilongjiang Provincial Crop Variety Approval Committee in 2002 by the Soybean Research Institute of Heilongjiang Academy of Agricultural Sciences. The first breeder was Researcher Du Weiguang, the patent number is CNA20020216.2, and the approval number is Heishendou 2002003.

[0074] The above-mentioned biological materials are available to the public from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences. These biological materials are only for the purpose of repeating the experiments of this invention and may not be used for other purposes.

[0075] The data in the following examples were processed using SPSS 11.5 statistical software. The experimental results are expressed as mean ± standard deviation. One-way ANOVA was used, and P < 0.05 (*) indicates a significant difference, and P < 0.01 (**) indicates a highly significant difference.

[0076] Example 1: Obtaining the soybean GmHSF35 gene

[0077] Transcriptomes of soybean varieties with high and low protein content were analyzed during the early and middle stages of rapid protein accumulation in seed development, identifying differentially expressed genes. Based on the soybean genome sequence, transcription factor binding elements contained in the 2.5 kb promoter regions of 11S and 7S soybean protein subunits were cloned and analyzed. Transcription factors binding to these elements were identified as candidate genes using a yeast single-hybrid system. These candidate genes were overexpressed in Arabidopsis thaliana to screen for genes involved in regulating seed protein content. Primers were designed to clone the target gene from HN44 based on our sequenced HN44 genome. All candidate genes were overexpressed in the model plant Arabidopsis thaliana for functional verification. The results showed that the protein content in Arabidopsis seeds overexpressing the candidate gene Glyma.17G174900 was significantly lower than that in the control, suggesting that it may negatively regulate seed protein content.

[0078] GmHSF35 gene cloning: Total RNA was extracted from HN44 seedlings, and the RNA was reverse transcribed using reverse transcriptase to synthesize cDNA. Based on the soybean genome sequence in PlantGDB and the full-length cDNA sequence of Glyma.17G174900 in the HN44 genome sequence we determined, primers were designed, and the primer sequences are as follows:

[0079] Glyma.17G174900-up: 5′-ATGTCACAGCAGCAGCGT-3′;

[0080] Glyma.17G174900-dp: 5′-CTAGACTAGGGTCTTCAATTCCTT-3′.

[0081] Using HN44 cDNA as a template, PCR amplification was performed using Glyma.17G174900-up and Glyma.17G174900-dp as primers, yielding a PCR product of approximately 0.9 kb.

[0082] HN44 leaf samples 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 to precipitate the RNA, which was then dissolved in water to obtain total RNA. 1 μg of total RNA was reverse transcribed using a Thermo Fisher Scientific kit according to the kit's instructions. The resulting cDNA fragment was then used as a template for PCR amplification.

[0083] The 50 μl PCR reaction system consisted 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, with the volume made up to 50 μl using ultrapure water.

[0084] 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℃.

[0085] A PCR product of approximately 0.9 kb was obtained. Sequencing revealed that this PCR product is sequence 1 (SEQ ID NO: 1, 849 bp) from the sequence listing, encoding the GmHSF35 gene. Alignment confirmed that Glyma.17G174900 is a member of the heat shock protein transcription factor (HSF) family, registered in GenBank as GenBank: NM_001353466, denoted as "heat stress transcription factor 35". GmHSF35 is known to be associated with stress tolerance, but its association with seed protein accumulation has not been reported. The amino acid sequence of this protein is sequence 2 (SEQ ID NO: 2, 282aa) from the sequence listing, as follows:

[0086] MSQQQRSTPAPFLTKTYLLVDDPATDDVVSWSEGGNTFVVWKHADFAKDLLPKYFKHNNFSSFVRQLNTYGFRKTVPDKW

[0087] EFANEYFKRGQTDLLAEIRRRKVVSPVTGKSTGGGVNISASHSGGDDMGSTSTGSMEAATAAGADISGENEKLKKDNEKL

[0088] SGELALAKKQCEELVAFLRDSLNVSPDVIERIIRQGTCVSSNNDAVRFDAIINNDLDDDENAVGECEPQQPPPKGDNGESLKLFGVWLKGQNGKENSIGKCRKRGRQDYPIAATAKELKTLV.

[0089] The genomic sequence of the GmHSF35 gene is sequence 3 in the sequence listing (SEQ ID NO: 3, 3841bp). Positions 1-368 of sequence 3 are 5'UTR, positions 369-578 and 2916-3554 are exons, positions 579-2915 are introns, and positions 3554-3841 are 3'UTR.

[0090] Example 2: GmHSF35 gene mutation regulates protein content in seeds

[0091] 2.1 Obtaining GmHSF35 gene mutants using gene editing technology

[0092] 2.1.1 Construction of gene editing vectors

[0093] 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.

[0094] A single-gene, dual-target knockout approach was adopted. The selected target sites were all located in the second exon region of the GmHSF35 gene, with target sequences T1 and T2 (nucleotides 248-270 and 319-341 of SEQ ID NO: 1, which correspond to nucleotides 2953-2975 and 3024-3046 of SEQ ID NO: 3). The positions of T1 and T2 in SEQ ID NO: 1 are shown below. Figure 1 As shown.

[0095] T1:5′-TCCTCGCTTGAAATACTCATTGG-3′;

[0096] T2: 5′-GTGACCGGAAAATCTACCGGCGG-3′.

[0097] Promoter selection: AtU6 derived from Arabidopsis thaliana was used to promote the T1 and T2 target sites.

[0098] Preparation of sgRNA expression cassettes containing target sites: The WMC024 vector was linearized by digestion with Bsa I enzyme. 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). The reverse complementary sequences (Anti-U6-T1, Anti-U6-T2) were synthesized, annealed to form double strands, and homologously recombined with the backbone linear vector.

[0099] The specific steps are as follows:

[0100] Sense-U6-T1: 5′-caatggtctcatgattgCCTCGCTTGAAATACTCATgttttagagctagaaata-3′;

[0101] Anti-U6-T1: 5′-tatttctagctctaaaacATGAGTATTTCAAGCGAGGcaatcatgagaccattg-3′;

[0102] Lowercase letters represent homologous arms, and uppercase letters represent T1 sequences.

[0103] Sense-U6-T2: 5′-ttggggtctctaaacCCGGTAGATTTTCCGGTCAcaatcactacttcgactctag-3′;

[0104] Anti-U6-T2: 5′-ctagagtcgaagtagtgattgTGACCGGAAAATCTACCGGgtttagagaccccaa-3′;

[0105] Lowercase letters represent homologous arms, and uppercase letters represent T2 sequences.

[0106] 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.

[0107] Vector linearization by enzyme digestion: 1-2 μg of WMC024 plasmid, 10×CutSmartTM 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.

[0108] Homologous recombination of the target sgRNA expression cassette with the WMC024 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 WMC024 vector, and 4.5 μL of annealed AtU6-T1-gRNA and AtU6-T2-gRNA. The reaction conditions were 50℃ 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 WMC024-sgRNA was obtained.

[0109] The recombinant vector WMC024-sgRNA is a recombinant plasmid obtained by replacing the fragment between 5′-cctcgcttgaaatactcat-3′ and 5′-tgaccggaaaatctaccgg-3′ in the WMC024 vector with the DNA sequence of SEQ ID NO: 4, while keeping the other sequences of the WMC024 vector unchanged. The recombinant plasmid is named recombinant vector WMC024-sgRNA.

[0110]

[0111] 2.1.2 Genetic transformation of soybean

[0112] The recombinant vector WMC024-sgRNA was transformed into Agrobacterium EHA105 competent cells (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 SciU S A. 2021 Jan, 12;118(2):e2013338118.doi:10.1073 / pnas.2013338118) to obtain Agrobacterium EHA105-WMC024-sgRNA.

[0113] The Agrobacterium EHA105-WMC024-sgRNA prepared above was used to infect the soybean variety Jack, resulting in soybean plants with the GmHSF35 gene edited. T0 generation seeds were harvested, and T1 generation seeds were obtained by self-pollination. T2 generation seedlings were then planted from the T1 generation seeds and subjected to the following tests.

[0114] 2.1.3 Screening for homozygous GmHSF35 gene mutations

[0115] Using the genomic DNA of the T2 generation GmHSF35 gene-edited seedlings obtained in step 2 as a template, PCR primers designed approximately 364 bp upstream and 247 bp downstream of the target sequence T1 were used to amplify the DNA sequence of approximately 671 bp near the target sequence and sequenced to detect the GmHSF35 gene editing method. The PCR identification primer set for the target site is as follows: 6220-F: 5′-GTAAGTTTCAATACGTTTCTGGTCA-3′, 6220-R: 5′-CCTCGCATTGTTTCTTCGCC-3′.

[0116] After successful sequencing, the gene editing methods were analyzed using the CRISPR target editing analysis 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 the sequences were passaged. The gene editing methods of the screened GmHSF35 homozygous mutants hsf35-1 and hsf35-2 are as follows: Figure 1 As shown.

[0117] Compared to the receptor Jack, the GmHSF35 gene in the hfs35-1 mutant genome has the following mutation: four deoxyribonucleotides GTAT are deleted in positions 255-258 of SEQ ID NO: 1 (corresponding to positions 2960-2963 of SEQ ID NO: 3), causing premature termination of GmHSF35 translation, thereby knocking out the GmHSF35 gene.

[0118] Compared to the receptor Jack, the GmHSF35 gene in the hsf35-2 mutant genome has the following mutation: an 80-nucleotide deletion exists at positions 255-334 of SEQ ID NO: 1 (corresponding to positions 2960-3093 of SEQ ID NO: 3) on both homologous chromosomes, namely the deletion of the nucleotide “5′-GTATTTCAAGCGAGGACAGACGGATCTTCTCGCCGAGATCAGGCGGCGGAAGGTGGTGTCTCCGGTGACCGGAAAATCTA-3′”, which causes premature termination of GmHSF35 translation, thereby knocking out the GmHSF35 gene.

[0119] Seeds from T2 generation plants (T3 generation) of mutants hsf35-1 and hsf35-2 were harvested for subsequent experiments.

[0120] 2.2 Phenotypic analysis of GmHSF35 gene CRISPR mutants hsf35-1 and hsf35-2

[0121] 2.2.1 Detection of GmHSF35 gene expression levels in hsf35-1 and hsf35-2

[0122] Total RNA was extracted from Jack and hsf35-1 and hsf35-2 seeds during mid-development and reverse transcribed. The cDNA obtained from the reverse transcription was used as a template. The primers were: HSF35-qF: 5′-CCAGCAACAATGATGCGG-3′; HSF35-qR: 5′-GTAGTCTTGGCGCCCTCT-3′. Real-time PCR was used to identify the expression level of the GmHSF35 gene.

[0123] 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 2 As shown, the relative expression level of GmHSF35 in Jack was approximately 1.028, while the expression levels in hsf35-1 and hsf35-2 were approximately 0.607 and 0.480, respectively. This indicates that the expression level of GmHSF35 in hsf35-1 and hsf35-2 was significantly decreased. Figure 2 ).

[0124] 2.2.2 Downregulation of the GmHSF35 gene increased seed protein content.

[0125] Seeds of hsf35-1 and hsf35-2 (seeds from T2 generation plants of the transgenic event in 1.2) and control Jack seeds were sown in greenhouse pots, maintaining an ambient humidity of 70-75% during growth. Greenhouse conditions: 16 hours of light at 11000 Lux and 8 hours of darkness; daytime temperature of 30-37°C and nighttime temperature of 25-28°C. Growth and development were observed. Seeds were harvested after 130 days. No significant differences were observed in the phenotypes of hsf35-1, hsf35-2, and control Jack during the growth and maturity stages.

[0126] After the above-mentioned potted soybean seeds matured, the seeds harvested from individual plants were dried at 37℃ for one week, and the protein content in the seeds of the recipient controls Jack, hsf35-1, and hsf35-2 was measured.

[0127] Total protein content determination in seeds: Seed protein was determined using a Thermo Flash2000 organic elemental analyzer (Molecular Agriculture Center Platform, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences). Mature soybean seeds were harvested, dried at 37℃ for three days, and then thoroughly dried at 65℃ for one day. 0.004 g of seeds were weighed for each sample and wrapped in a tin bag. Based on the Dumas combustion method, the sample was fed into the combustion reaction tube via an autosampler. After combustion, further reaction was performed to determine the nitrogen (N) content in the sample. The measured value was multiplied by a conversion factor of 6.25 to obtain the crude protein content of the sample.

[0128] Seeds from 10 plants of each line were taken, and the experiment was repeated three times. The results were taken as mean ± standard deviation.

[0129] Seed protein content statistics are as follows Figure 3As shown, the protein content in the seeds of the recipient controls Jack, hsf35-1, and hsf35-2 was 32.5±1.4%, 35.1±0.9%, and 35.4±1.2%, respectively. The mutants hsf35-1 and hsf35-2 showed increases of approximately 8.0% and 8.9% compared to the control Jack, respectively, which were highly significant.

[0130] The above results show that, in three biological replicates of 10 individual plants each from the control and the two mutant greenhouse potted plants, the protein content in hsf35-1 and hsf35-2 seeds was significantly higher than that of the receptor Jack. This indicates that GmHSF35 negatively regulates the protein content in soybean seeds, and reducing the expression level of the GmHSF35 encoding gene can significantly increase the protein content in soybean seeds.

[0131] 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 regulating the expression of the gene encoding said protein, or a substance regulating the activity or content of said protein, in any of the following: A1) Application in regulating protein content in plant seeds; A2) Application in the preparation of products that regulate the protein content of plant seeds; A3) Application in cultivating plants with altered protein content in seeds; A4) Application in the preparation of products from plants whose protein content has been altered in the seeds; A5) Applications in plant breeding; The protein is GmHSF35, and is any of the following proteins: B1) A protein with the amino acid sequence SEQ ID NO: 2; B2) A protein with the same function as the amino acid sequence shown in SEQ ID NO: 2, but with amino acid residue substitution and / or deletion and / or addition. B3) Proteins that share more than 80% identity with the amino acid sequence defined by B1) or B2) and have the same function; B4) A fusion protein obtained by attaching a tag to the end of any of the proteins defined in B1)-B3).

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 regulation is at least one of the following six types of regulation: C1) Regulation at the transcriptional level of the encoded gene C2) Regulation that occurs after transcription of the encoded gene C3) regulates the RNA transport of the encoded gene. C4) regulates the translation of the encoded gene. C5) regulates the degradation of the mRNA encoding the gene. C6) regulates the post-translational regulation of the gene.

4. The application according to any one of claims 1-3, characterized in that, The substance regulating the expression of the gene encoding the protein or the substance regulating the activity or content of the protein is a biological material related to the protein in the application of claim 1 or 2, wherein the biological material is any one of the following: D1) Nucleic acid molecules that inhibit, reduce, or silence the expression of the protein-encoding gene as described in claim 1 or 2; D2) An expression cassette containing the nucleic acid molecules described in D1); D3) A recombinant vector containing the nucleic acid molecule described in D1), or a recombinant vector containing the expression cassette described in D2); D4) Recombinant microorganisms containing the nucleic acid molecules described in D1), or recombinant microorganisms containing the expression cassette described in D2), or recombinant microorganisms containing the recombinant vector described in D3); D5) A transgenic plant cell line containing the nucleic acid molecule described in D1), or a transgenic plant cell line containing the expression cassette described in D2); D6) Transgenic plant tissue containing the nucleic acid molecules described in D1), or transgenic plant tissue containing the expression cassette described in D2); D7) Transgenic plant organs containing the nucleic acid molecules described in D1) or transgenic plant organs containing the expression cassette described in D2).

5. The application according to claim 4, characterized in that: D1) The nucleic acid molecule is a gRNA that targets the protein-coding gene described in claim 1.

6. A method for increasing the protein content in plant seeds, 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, to increase the protein content in the plant seed; the target plant contains the gene encoding the protein described in claim 1 or 2.

7. A breeding method for cultivating plants with increased protein content in their seeds, characterized in that: The method includes step M, which involves inhibiting, reducing, or silencing the activity and / or content of the protein described in claim 1 or 2 in the target plant, and / or inhibiting, reducing, or silencing the expression level of the gene encoding the protein described in claim 1 or 2, to obtain a plant with increased protein content in its seeds, wherein the protein content in the seeds of the plant with increased protein content is higher than that of the target plant; the target plant contains the gene encoding the protein described in claim 1 or 2.

8. The method as described in claim 6 or 7, characterized in that, Step M includes introducing a gene knockout vector into the target plant with nucleotides 2953-2975 and / or nucleotides 3024-3046 of sequence 3 in the sequence listing as the target sequence.

9. The method as described in claim 6 or 7, characterized in that, The target plant contains the gene encoding the protein of claim 1 or 2, and step M includes performing any of the following operations on the genome of the target plant: E1) Delete the four deoxyribonucleic acid GTATs between positions 2960-2963 of sequence 3 in the genome of the target plant; E2) Delete 80 deoxyribonucleotides between positions 2960-3093 of sequence 3 in the genome of the target plant.

10. The application according to any one of claims 1-5, and / or the method according to any one of claims 6-9, 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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