Application of soybean GmCDC7 protein and coding gene thereof in regulation and control of soybean grain shape and grain weight

Abstract

The invention discloses application of soybean GmCDC7 protein and a coding gene thereof in regulation and control of soybean grain shape and grain weight. The GmCDC7 gene encodes a plant germination-like protein, and the amino acid sequence of the GmCDC7 gene is as shown in SEQ ID No.2. According to the invention, gene editing is carried out on a GmCDC7 gene of a receptor plant soybean variety Jack by using a CRISPR-Cas9 technology, and a homozygous mutant of the GmCDC7 gene is obtained. Compared with a wild type, the soybean seed hundred-grain weight, grain length, width and thickness of a GmCDC7 gene mutation plant are obviously increased, which indicates that the GmCDC7 gene negatively regulates the grain shape and grain weight of the soybean grain. The invention discloses the biological function of the soybean GmCDC7 gene for controlling the soybean grain shape and grain weight for the first time.

Description

Technical Field

[0001] This invention belongs to the field of mutation or genetic engineering, specifically relating to the application of soybean GmCDC7 protein and its encoding gene in regulating soybean seed shape and quality. Background Technology

[0002] Soybeans (Glycinemax), as both a food and oilseed crop, are an important food ingredient and livestock resource for countries around the world, occupying a significant position in global food consumption. Soybeans have a wide range of uses; they can be eaten directly, processed into various soy products (such as tofu, soy milk, and dried tofu), extracted for soybean oil, brewed for soy sauce, and used to extract protein.

[0003] Soybean yield is composed of factors such as plant type, pod setting rate, number of pods and seeds, and 100-seed weight, among which seed weight has the highest heritability. Seed weight is an important indicator in seed crop production and one of the important agronomic traits affecting crop yield. Plants can increase yield by increasing seed weight. Most wild soybeans have a 100-seed weight of only about 2 grams, while cultivated soybeans generally have a 15-22-gram weight, and can reach up to 25 grams, showing a significant difference.

[0004] Researchers have successfully cloned several genes related to soybean grain traits and verified their functions through genetic transformation and expression pattern analysis. For example, the GmSWEET10 gene plays a crucial role in controlling soybean seed development and oil accumulation; knocking out or increasing the expression of this gene can significantly affect soybean grain weight, oil content, and protein content (Wang et al., 2020); POWR1 regulates protein and oil content, while also affecting grain weight and field yield (Goettel et al., 2022). Currently, there are relatively few known genes regulating soybean grain weight, and the molecular research foundation is weak. The molecular genetic basis and regulatory network of soybean quality are still unclear. Summary of the Invention

[0005] The technical problem to be solved by this invention is how to improve the grain traits of plants, especially soybeans.

[0006] To address the above problems, the present invention provides a method for improving plant seed traits, the method comprising improving the seed traits of a target plant by gene knockout, wherein the gene knockout is the knockout of the gene encoding a target protein in the target plant, and the target plant contains the gene encoding the target protein;

[0007] The target protein is GmCDC7, and is any one of the following:

[0008] A1) The amino acid sequence is that of the protein shown in sequence 2.

[0009] A2) A protein obtained by substituting and / or deleting and / or adding amino acid residues of the protein described in A1), which has more than 80% identity with the protein shown in A1) and can regulate plant grain traits.

[0010] A3) A fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of A1) or A2).

[0011] The present invention also provides a method for cultivating plants with improved seed traits, comprising obtaining plants with improved seed traits by gene knockout, wherein the target plant contains the gene encoding the aforementioned protein.

[0012] 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.

[0013] In the above-mentioned proteins, identity refers to the identity of the amino acid 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 program, 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 an identity search on a pair of amino acid sequences, the identity value (%) can then be obtained.

[0014] In the aforementioned proteins, the 80% or more identity can be at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 95%, 96%, 98%, 99%, or 100% identity.

[0015] The protein described above is sequence 2 (SEQ ID No. 2) in the sequence listing. It consists of 943 amino acid residues and is named GmCDC7 protein or protein GmCDC7. Its encoding gene is the GmCDC7 gene.

[0016] The gene knockout includes introducing a gene knockout vector into the target plant targeting nucleotides 422-444 of sequence 1 in the sequence listing.

[0017] The protein mentioned above is derived from soybeans.

[0018] In the above method, the genome of the target plant contains a DNA molecule whose nucleotide sequence is sequence 1 in the sequence listing, and the gene knockout may include performing any of the following operations on the genome of the target plant:

[0019] B1) Delete nucleotides TCA from position 429 to 431 of sequence 1 in the sequence listing of the target plant.

[0020] B2) Delete nucleotides 429-432 of sequence 1 in the sequence listing of the target plant.

[0021] B3) Delete nucleotides CCGTCA from position 426 to 431 of sequence 1 in the sequence listing of the target plant.

[0022] B4) Delete nucleotides CGTCAGCGCCGTCTTCGCCCTC from position 427 to 448 of sequence 1 in the sequence listing of the target plant.

[0023] This invention also protects the application of biological materials, which may be any of the following:

[0024] D1) The application of the biomaterials described herein in improving plant seed traits and / or in preparing products that improve the seed traits of the target plant.

[0025] D2) The application of the biomaterials described therein in plant breeding and / or the preparation of plant breeding products.

[0026] The indicators for plant breeding include the seed traits of the plant, and the purpose of plant breeding includes improving the seed traits of the plant.

[0027] The biological material is a protein, a substance that regulates the expression of the gene encoding the protein, or a substance that regulates the content of the protein.

[0028] The protein is GmCDC7, and is any one of the following:

[0029] The amino acid sequence of E1 is the protein shown in sequence 2.

[0030] E2) A protein obtained by substituting and / or deleting and / or adding amino acid residues of the protein described in E1), which has more than 80% identity with the protein shown in E1) and can regulate plant grain traits.

[0031] E3) is a fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of E1) or E2).

[0032] 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.

[0033] In the above-mentioned proteins, identity refers to the identity of the amino acid 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 program, 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 an identity search on a pair of amino acid sequences to calculate the identity value (%), then the identity value can be obtained.

[0034] In the aforementioned proteins, the 80% or more identity can be at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 95%, 96%, 98%, 99%, or 100% identity.

[0035] The protein described above is sequence 2 (SEQ ID No. 2) in the sequence listing. It consists of 943 amino acid residues and is named GmCDC7 protein or protein GmCDC7. Its encoding gene is the GmCDC7 gene.

[0036] In the above text, the substance referred to is any one of the following:

[0037] F1) Nucleic acid molecules that inhibit, reduce, or downregulate the expression of the genes encoding the aforementioned proteins.

[0038] F2) expresses the gene encoding the nucleic acid molecule described in F1).

[0039] F3) contains the expression cassette of the gene described in F2).

[0040] F4) a recombinant vector containing the gene described in F2), or a recombinant vector containing the expression cassette described in F3).

[0041] F5) Recombinant microorganisms containing the gene described in F2), or recombinant microorganisms containing the expression cassette described in F3), or recombinant microorganisms containing the recombinant vector described in F4).

[0042] F6) A transgenic plant cell line containing the gene described in F2), or a transgenic plant cell line containing the expression cassette described in F3), or a transgenic plant cell line containing the recombinant vector described in F4).

[0043] F7) Transgenic plant tissue containing the gene described in F2), or transgenic plant tissue containing the expression cassette described in F3), or transgenic plant tissue containing the recombinant vector described in F4),

[0044] F8) A transgenic plant organ containing the gene described in F2), or a transgenic plant organ containing the expression cassette described in F3), or a transgenic plant organ containing the recombinant vector described in F4).

[0045] In the above applications, the nucleic acid molecule described in F1) is an sgRNA that targets the protein-coding gene described above.

[0046] The transgenic plant is a plant obtained through biological methods such as recombinant DNA technology in genetic engineering.

[0047] Furthermore, in the biological material, the recombinant microorganisms mentioned in F5) can specifically be yeast, bacteria, algae, and fungi.

[0048] Furthermore, the recombinant microorganism may be Agrobacterium. Specifically, Agrobacterium is EHA105.

[0049] Furthermore, in the biological material, the plant tissue described in F7) may be derived from roots, stems, leaves, flowers, fruits, seeds, pollen, embryos and / or anthers.

[0050] Furthermore, in the biological material, the transgenic plant organs described in F8) can be the roots, stems, leaves, flowers, fruits, and seeds of the transgenic plant.

[0051] In the above text, the improvement of plant grain traits can be selected from at least one of the following: H1) increasing the grain weight of plant grains, H2) increasing the grain length of plant grains, H3) increasing the grain thickness of plant grains, and H4) increasing the grain width of plant grains.

[0052] The particle weight can be the weight of 100 particles.

[0053] The plant mentioned above may be any of the following: J1) a dicotyledonous plant, J2) a plant of the order Rosales, J3) a legume, J4) a plant of the genus Glycine, J5) soybean.

[0054] This invention utilizes CRISPR-Cas9 technology to edit the GmCDC7 gene in the recipient soybean variety Jack, obtaining a homozygous mutant of the GmCDC7 gene. Compared with the wild type, the GmCDC7 gene mutant plants showed a significant increase in the 100-seed weight, and improvements in seed length, width, and thickness. This indicates that the GmCDC7 gene negatively regulates soybean seed shape and weight. This invention reveals for the first time the biological function of the soybean GmCDC7 gene in controlling soybean seed shape and weight traits, providing new germplasm resources for genetic breeding work and playing a positive role in improving soybean varieties. Attached Figure Description

[0055] Figure 1 This is a homozygous mutation of the GmCDC7 gene in T1 generation plants.

[0056] Figure 2 Phenotypic identification of soybean seeds from GmCDC7 gene mutant plants. A shows the phenotypic diagrams of seeds from GmCDC7 gene mutant plants and wild-type plants; B shows a comparison of seed length between GmCDC7 gene mutant plants and wild-type plants; C shows a comparison of seed width between GmCDC7 gene mutant plants and wild-type plants; D shows a comparison of seed thickness between GmCDC7 gene mutant plants and wild-type plants; E shows a comparison of the weight of 100 seeds between GmCDC7 gene mutant plants and wild-type plants. Detailed Implementation

[0057] 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.

[0058] 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.

[0059] The following examples use SPSS 11.5 statistical software to process the data. The experimental results are expressed as mean ± standard deviation. One-way ANOVA test was used. P < 0.05 (*) indicates a significant difference, P < 0.01 (**) indicates a highly significant difference, and P < 0.001 (***) indicates a highly significant difference (please change according to the actual situation).

[0060] Soybean material: Soybean “Jack” was kindly provided by Researcher Wensheng Hou of the Institute of Crop Science, Chinese Academy of Agricultural Sciences, and is “the soya bean cultivar Jack” in the following literature: Cai Y, Chen L, Liu X, Guo C, Sun S, Wu C, Jiang B, Han T, Hou W. CRISPR / Cas9-mediated targeted mutagenesis of GmFT2 adelays flowering time in soya bean. Plant Biotechnol J. 2018 Jan; 16(1):176-185. doi:10.1111 / pbi.12758. Epub 2017 Jun 20. PMID:28509421; PMCID:PMC5785355.

[0061] References for strain EHA105: Komari T, Halperin W, Nester EW. Physical and functional map of supervirulent Agrobacterium tumefaciens tumor-inducing plasm idpTiBo542. J Bacteriol. 1986 Apr; 166(1):88-94. doi:10.1128 / jb.166.1.88-94.1986.PMID:3957875;PMCID:PMC214561.

[0062] The pGES201 in the following examples is the third to fourth segment from the bottom of the right column on page 728 of the following literature. Its full sequence can be found in the Supporting information section of that literature. Supplemental Figure 1: The sequence of pGES201 vector: Mengyan Bai1, Juehui Yuan1, Huaqin Kuang, Pingping Gong, Suning Li, Zhihui Zhang, Bo Liu, Jiafeng Sun, Maoxiang Yang, Lan Yang, Dong Wang, Shikui Song and Yuefeng Guan. Generation of a multiplex mutagenesis population via pooled CRISPR-Cas9 in soya bean. Plant Biotechnol J. doi:10.1111 / pbi.13239.

[0063] The aforementioned biological materials are available to the public from the Institute of Botany, Chinese Academy of Sciences. These biological materials are intended solely for repeating the relevant experiments of this invention and may not be used for any other purpose.

[0064] Example 1: GmCDC7 gene knockout

[0065] 1.1 Preparation of GmCDC7 gene knockout vector

[0066] Gene editing target site design was performed on the GmCDC7 gene in the soybean variety Jack using the online CRISPR-2.0 website. The target sequence is 5'- CCC TCCGTCAGCGCCGTCTTCGC-3' (bases 422 to 444 of SEQ ID No. 1, and bases 214 to 236 of SEQ ID No. 3).

[0067] The soybean GmCDC7 genome sequence (SEQ ID NO.1, 7512bp) is sequence 1 in the sequence listing, as follows:

[0068] 5'-GGAAACTTCATCGTATTCTCTCCAAAAAAGAAGCGGGAAAATTCACCCAAAACACAACACCCTCAAAAAT

[0069] GGCAAAATCCAAAACACTCTTCGGTCTTCTCTTCACTTCCAGTACTTCCCGCTCCTCTTTCTCCCTTTCTCTCT

[0070] CTCTCTAGACCTGGTCGCATCGCAGATTCGCAAGCACACACACTTCAGATCCGAATCTCGATCCATGGCAGAGT

[0071] CCGAATTCGAACCAAACCGAGTCCATGACCTCGAAGAGAAATCGTGGCACCTCCTCGCGCTCCTCTTCCGAATC

[0072] GGCCACGCCGTTTACCCGCAACGCCTCGCCGCGCAGTGCCGTTTGTTCGCCGCCTCGCCTGACTTCGTCTGCTA

[0073] CGTCAGCACTCTCCCCGGCTCGCCGCTCTCCGTCACCGACAACGGGCTCGTCACGCCCTCCGTCAGCGCCGTCT

[0074] TCGCCCTCGGAAGCTTCTTCTCCCTCCGCTTCTCGCCGCCGCAGACGCACCGTTTCAGGAAACGCAAGTTGCTT

[0075] TTCGATTCCGCCGAAGGTTCGTTTTTCTTTTTCTTTTTCTTCAATTTTAATTCTGATTTTTTTTATTTTTAATT

[0076] TCGTGGTTGATTTGATTTGATTTTGGTGAAAGCAGATGGAAGAGAGCGCAAGAGGTTAGCGATTCGTCATGGAC

[0077] TTCGAGAATTTTCGTTCCAGGTAGTCAGAAGCCAAAAATTCTATAAGTTAGCAGTAAAACTAGAAATTTATTTA

[0078] TCTGATACAAGAGCTACTTAGAGTTGTTTGAACTCAAAAATCAATTTTGAACCTAAAATCAATTCTTCAATATG

[0079] GAATCAAGCATGTTAATATTTACTTATAATCAAGGTAGCTGGTACTTTAACATTATTTTTAGAATGATCCGACATG

[0080] AATTCAAAAACTTTACTTTTTTGCTTATTGATACTTATAGAACGCGGAATCATACTTTTATTTGGCTTTCTTTC

[0081] CCATGTATAAAGGAAAACTTTTGTTTGGCTAGTGTGCGTGCCTTTGTATGTGACGGCGACAGTAGCTTGATTAT

[0082] GCATGAATTTCTTTTTTCTTGCATTTAGGATGCGTTATTATATAGTCTAGTGAAAGGAATAGAAATATAACCGT

[0083] TTAGATTGGAGTGACCATGATTATTATTCGTAAATGTCAATTTCTGAGGGTATAAGGTTGCATTTCGTTTCATC

[0084] TTAGGCATGTACTGGCAGTTTGTTTGAATGATAAAATAATTTAATGCATAGTGAATCACAATGATGTTTAGCCA

[0085] TACCTGTGTTAATGAACTTCATGAAACTTGGTGCCACCATGTACTGGCAGTTTGTTTGAATGATTTAATAATTT

[0086] AATGCATAGTCGAATATAATTGATTTTGTTTCTATAGCTAGTGTGTAGCTGTGATATTCGACAGTGGCACGCAC

[0087] TTCATTGAGAATGATTTGCATTTAGGGTGCATTACAGTCAGGTGGAACAGAAAGCTATAGAAATAAAATCAATT

[0088] TTATTCAGCTTTTTATTATTAATATATAAATAAAGTAGTAAATATCATTTCTGAGGCATAGTTTGGTTGCCTT

[0089] TCATTTGAGTTTCTTCTAAGAAAAAAAATCAATTAAAAATCTGATTAAATTATGTGATAATCAGATTTATTATT

[0090] TTTATTTTAAGAAAAAAACTAAAATGATGTTAGTTTTAGCCCTTTTCTTGTGAGTTATAATAAGTAACGTATGA

[0091] AATAAAGCCTACATTATAAAGCGTTTTTGTTTTTTCAAAGCTTAAACAAATGCATCTTTAATCAACCAACCTTAT

[0092] AGAGTGACATATATCTTTTATTTAGCCATCATTGTGTTTCGTGCTGAATATGGTGCTAAGCTTTGGGAATTTTA

[0093] TTTGGCGTGCAGAGTTTTGCTGATGCTGCAGAGGCTCTGATGAGAAGCAATTTCCCTGTAATAAAATTTGAATC

[0094] ACAGAATATAGGTAGTGGAAAATTTTGTATTCCTCTTGCGTATTGATAAAAATGAAGAGTGTTCTGGTTGTCCAA

[0095] TGTCAAATTTTGAACATCGGGGAGCCAATAATGATGCCAGCACAACCATGTCTAATGGAGAGTTTCTAAATCT

[0096] ATTATCGAAGTGCTGACCAAAAAAATATCCATTATCAAAACAAGTTACTTGAACTGTCCGAAGCTAGTGGACAA

[0097] AAATAGATTCATGGAATGCAATCTCTTTGTTGTGGATCCCCCATGCAAAGAGGGAATAGTTAACAACCTTGGAA

[0098] TTGGGAAGAAAATAGATTATTTTGACACATTTACGCATGACTGTGCTGAACAAAACAGTATTCATCATGTGGAT

[0099] GAAGATGGTATTGGTAAAAGTAATACTTGCAAAGATCAACCAAGAGAACCAAATGAAGATGATGAGGTGGAAAG

[0100] TGGTTCAAAGAAAGGGCTGATTGATTCTGGCACAAATAGAGTCAAGGAAGATGTTGCTCAGAGGGTCAATGCTG

[0101] CACTATGTGGAGAAGCATTGACTAATGGTTTGGAACAAAAGAACCATGTCCATGCAATGAATTTGGAAAAGGAG

[0102] AGTGAGAAAACACAGGGACTAAGTCTACTAATAAAATATCAAATTCCTCTTCAACTCCTAAGCGACCTTTGAA

[0103] ATCTTCTAGCATATTAAAGGGAGGACAGAAGAATGATCTCCACCCCAAGTCACAAATTCTTAAAGAGTCATTAG

[0104] TCAGTAATAAATTTGGTAATGTTCCAAAAAATGTTGATCAACGTAAAAATGAACAGAATCTTACAGCAAGGAAG

[0105] CAAAATCATAAAGAGAACATGGCTGGAAATATTGCCACTACTACTAAGGTGAGCAGCTTACACTAAGCTGTAGT

[0106] TCGAAATAGCCTGTGCTTTGAGAAGATGTTTTTTTAGATCATTTTGACTGTTTGATAAGAGGCATAACTTATG

[0107] TTCACTTTATCCATTTTGGTTATTTTGTTGAATCAGGTGGAGAAAAGAGCATATCCATCATTTGAAGCTTTTAC

[0108] AATAGAGGAAGAAGAAGGTTCAGGTATTTGGATTCTTTCTTTCTCCTCAACTTCCTTCTTGATGAAATAAATAT

[0109] TTAAATTGCATTGACATTTCTTATTTTTTTTCATATAGGTGGTTATGGCACCGTTTACCGTGCTCAAAGAACTA

[0110] CTGATGGAAAACGGGTTGCAATAAAATGTAAATGACACCTATCTTTTATATTAATTCTGTGCTGTGTGTTTAGT

[0111] GTTATAATTAAGAAAAGGAAAATGGGTAGTGTCCATTATTTGCACAAAATTAGAAAGTGAAGCTACTTATTTGT

[0112] CTTCAAAAATTCAGTTCTTCTTGTGTTCAGTTTCACACTTACAGTAGAAACTGGAAGCAATTCTTTGTATGTAA

[0113] TCATGTTGATATTACTAATATTAATTTACTTTATGATTGGCGTAATGGCTTTTAAGAAAATTTTGATATTGATGC

[0114] TCCAATATATATACATGTGATTTTACAGGTCCTCATACTAATGCTCATAAAAACCATGTAAATAATGAACGGAG

[0115] TATGCTCGAGCGTTTTGGGTAAGTATAAGACATAATGGTACAATATTTGTTATATTTAAGCTGCAATGCATTCT

[0116] GTTATTTGTTAATATTTTTTTCTCATGTGACAGTGGTAAAAACTATATAATAAGGTATGAGGGCTCTTTTAAAA

[0117] ATGGCAACAGCGACTGCTTTGTTTTAGAACATGTTCACCATGATAGACCTGAGGTAATTGCTGAAGCTGTCTAT

[0118] AATTTATATTTTCTTCATCAGAAAAACAATATCAGTTTCTGTTTCTACATATAATTTTTTTTCCAATTTAATCA

[0119] ATGAATCAAATTCAGGAGATGAATTCCCCTTTTATAGCCTTTACCAAAGTTAAACTGGTTAGCTCTTGAGAAGA

[0120] ACCC TACATTCTCTGAAAT TCCCTCTTTAGTAGAGCTAATTTTACTTCTTGATCTTTAGGTTCTGGAAAAGATA

[0121] TGCAAGAAATGGATTTCTTTTTCAATGTTTCTTATTTTAATTTAATTTGGCTCATGAGAGTACTCTGACCTGCT

[0122] CTACATTCATGTTTAATGGAGATAATTTTCCCTTCTCAATTTGTAGGTTTTGAAAAAAGAAATTGATATAGTTC

[0123] AGCTTCAATGGTACGGGTATTGCATGTTCAGGGCCCTTTATTGCTTGCACAAAGAGGTATTTCCCCCCTTGCAT

[0124] GATCAGAGTCTCATGTGACTGACTAAAAAGACAACAAGTACAATTGTTTTGAGTAATTTGAATTCTATTGTAGG

[0125] GAGTTGTTCACAGAGACATTAAACCTGGAAACTTCCTTTTCTCTCGAAAGCTAAGCAAAGGCTATCTTATTGAT

[0126] TTTAACCTTGCCATGGTTAGATTGCCTCACTTTCTTCTTCTTATCAAGAGTTTTGACTGATTCTTTTAATATAA

[0127] ATACTGATGAATTTTGATATTTTCTTTCTAGGATTTAAAGCAGAAGCACAACACATCGGAAGTAAGATTTCTTTTC

[0128] ACTTATCCGTTGCTTATTCTTTTGAGAAATAGAAAAACTGGCTGAATATTTGGTTTTCTTATGACACTTGTAAT

[0129] TTTCATAAAATAAGACATGGTGCCTCTTTGGTAACTTGGTTAACTAATTTCAAGATTGAAGACAACACACTCTT

[0130] AGAACAAAACAACATTTTTCTTGCTTCTAAGATTCTACCTGAATTATATTTATTTCTCTCAAATATTCCTTAGT

[0131] TAAGTTTTACTGTCAAATGATATTCATAATATAATCTGCTTAATTAGTTTAGTCAATTGCACATCTTTTCAGGT

[0132] AAATCTAAACCAAGCCATGATGCAGCATCCAATATTGTTTCTTTCTCTTCTGGTTCTGCCCCTCTGGTCCGAGA

[0133] CAAGAACCTTGGAGGCAGCAAGTCTTTAACATCCAATAAAAGGGCTTTGGCAGATTATAAGAATTATTCTGAAC

[0134] TTAATAGGCATGTAAAGCAAAAGGATTGTACCGGTCCTCTGAAAAATTGTCCTGATAAGGCTGGTGGGAGTTTT

[0135] CTTAGAGCACAAGGAACAGATGGCTCTGGTGTAACTTCTGCGAAAGATCCTAGCACTAGGACTGCTTCTGCAGA

[0136] GAGGCTCAGGGAACCTTTACCTTCCCATGGAAGAAAGGAGCTTATCAGCTTTGTGAATACCATGAAATGTGCAA

[0137] ACAACAGTTCAACAATAGGCCCTTCTTCACAAAGGAAAAGGGTTACTGCTCCCTCAAGCAAGGTAGATGGCAAG

[0138] ATTTTTAATATTACTCCGATGCCTTTGCATTCATCTACTGTTGGTGGGGGATTAATGAGAAGCAAAGGTATGAC

[0139] TCAAATTTTATAACTTCTAAGTTCCTAGGAGTTTTATTGCTACCTTTATCTTATTAACTCTTGGTGTGTTTTCC

[0140] AGGTGATGGAAAGAAAAAAGAAGGTTCATGTGTTGGAACCAAAGGATTCCGTGCTCCGGAGGTAAGATCTTGCT

[0141] GTCACTTCTATTCACTTGCATCTTTTTACCCATTTAAAGGCTCAATTTATGCTGATCCAAATAGAGATAGTCTA

[0142] TAATGAATGTTGACATAGAGAGTGCCATTTTTAGGTGCTTGTTGAAAAAAGAGAATTAGAAACATTACTATTAGC

[0143] ACCTGGAATGACAATTTTATACTTCTGTGGAAGTTAAAAGTGAAGCAGTTGCCTTCCCTAAACCTAAAACAAAT

[0144] CACTATTCATACTTTGGGATACACTATTTCCTTTAGCCCATGTACGATTTATTGGAATAGTATTCTGAGTTAGC

[0145] ATAAGTTTCAACTGGTTACAATCTAGTTTCCCAGTTGCAGTATAGGTTTCAGTATCCCATATTTACCATTCCGT

[0146] TTCATTTGTGGCAAATTGTGGTCTCCTGCTGACTAAAGAAACTTAATTATGAGAATTTTACTTTGAAGGATA

[0147] CCCTTTCACGGTCTGTATGGCCAACTGAGATATCCATGATTTGAATGATTTAATTGTACTAGATAACTAGTCTG

[0148] AAATATATTTCACCCTATGAGACTATTGGTCTCTTCTACTTTTTCAGAATTTAAATGAAACTATCTTTGGGCAG

[0149] GTAAAGAGAGGAAATAAACAATGTAAGATTCTGATATTTAATTCTTAAGTTTACATAGCGCATTCAAGCTGTAAA

[0150] GGCTTCATTATAAAAGCTAAATTTATGGTCTATACCTGATGGGACATTTTAAGTTTGTTACCTAGAGAAGCTAT

[0151] TTCACTGTCCAATAATTTAATTTATGGCTTGTTTTTCAGTAGTTTCAATTCAAGGTTCTACCTTTTCTTCAGGT

[0152] TTTGTTAAGGTCTCAGCATCAGGGACATAAGATTGATATTTGGTCAGCTGGAGTCACTCTACTCTACATGGTGA

[0153] TCGGGAAGACTCCTTTCACTGGCGACCCAGAACAGTAAAAACCACCTGCAAAATTCATTTGTGATGATTTTATT

[0154] TGTTTCGTTGTTTGTCTGTCCATGCTGACAGTTTTGTTGCAGGAATATAAAAGAAATTGTTAAATTGCGGGGCA

[0155] GTGAAGAGTTTTGGGAAGTGGCCAAGCTACATGACCGTGAATTATTTTTCCAGTGGTAATTTCTTTTATTCCT

[0156] TATTTTTAAAACACTTAATATTGAGAAAAAAATATTAAACTGAAATAATCTTCGAATTAATTAAAATGTTTATT

[0157] ATTCTATTAAGTTTCATGCAAAAATATATTTATAAATGAATATTCTTTTATCTGTCTTGTTAGAGATTATTGG

[0158] TTAAACTTCATAAGTCCAGTTGTATAGTGTTTACTAGTAAATAAATTGTCCGTAAATTAATTCTATGATAATGT

[0159] TTAAAGATGATGGTTGCTTGCACAAACTTTATTTTGGTTTTTAACAGTTGAAATTGTGATTTTAATTCATTAGC

[0160] TCTGAACTCAACTGAGGTTACTACAAAAGAAAAGAGCTCTTATTTTTTACTAATATAGTAAATGAAGCAAAATG

[0161] ATTAATGATTGTACTAATCACCTTAGTTAAATTCGTTGAGTTGCAGGAGTTACTTGATGACCGATACTTAC

[0162] AGTCATGGGACTTAGAAGGCTGGTGCAAGATTCACACAAAGAGACCAGAGTTTCTTGAGCAGATCCCGAAATCG

[0163] CTGTTTGATTTGATAGACAAGTGTCTAACAGTTAACCCAAGAAATAGGCTCAGTGCTGAAGATGTTCTAAGGCA

[0164] TGAATTCTTTGACTCACTGCATGAGTCCCTGAGGAAGCAAAGGATGCTTCATCGACATCGAGCTCTCAGGTCGG

[0165] ATGCAGCAGCTTCCAGAGCAATCTGACATCAGTTTCATCAAGTGCATGTACTTTTGGTAGCAACTGACCACATA

[0166] AGTAGTTGTTACTGTATATTTTTTGAGGTGAAGGTGGTTAACACATAAGGGGTTTCCTTACCTGTTAGTGATG

[0167] GCTTTTTACTGTATATATAACTATATCAGAGAAACCTAGCAGTGTAACTTGTAAGTTGTGTTAAAATTTAGAACT

[0168] TTAACATATTTTTGTATTTATTATTTTATTGCTGTAAAGTGTTTTTCTCATTTATTTTTTTAGGTTTCCAACTAATTATGTTTGTTCAAACTTTTTATAATCTTTCATCTTATTTAA-3'.

[0169] Positions 1-208 are 5'UTRs, and positions 7201-7512 are 3'UTRs; positions 209-530, 624-682, 1859-2782, 2919-2979, 3069-3131, 3429-3492, 3582-3675, 4039-4122, 4214-4303, 4394-4421, 4731-5243, 5327-5385, 6211-6320, 6403-6490, and 6928-7200 are exons; the rest are introns.

[0170] The amino acid sequence of the protein GmCDC7 encoded by the soybean GmCDC7 gene is sequence 2 (SEQ ID NO. 2, 943aa) in the sequence listing, as follows:

[0171] MAESEFEPNRVHDLEEKSWHLLALLFRIGHAVYPQRLAAQCRLFAASPDFVCYVSTLPGSPLSVTDNGLVTPSV

[0172] SAVFALGSFFSLRFSPPQTHRFRKRKLLFDSAEDGRERKRLAIRHGLREFSFQSFADAAEALMRSNFPVIKFES

[0173] QNIGSGNFVFLLRIDKNEECSGCPMSNFEHRGANNDASTTMSNGEVSKSIIEVLTKKISIIKTSYLNCPKLVDK

[0174] NRFMECNLFVVDPPCKEGIVNNLGIGKKIDYFDTFTHDCAEQNSIHHVDEDGIGKSNTCKDQPREPNEDDEVES

[0175] GSKKGLIDSGTNRVKEDVAQRVNAALCGEALTNGLEQKNHVHAMNLEKESERNTGTKSTNKISNSSSTPKRPLK

[0176] SSSILKGGQKNDLHPKSQILKESLVSNKFGNVPKNVDQRKNEQNLTARKQNHKENMAGNIATTTKVEKRAYPSF

[0177] EAFTIEEEEGSGGYGTVYRAQRTTDGKRVAIKCPHTNAHKNHVNNERSMLERFGGKNYIIRYEGSFKNGNSDCF

[0178] VLEHVHHDRPEVLKKEIDIVQLQWYGYCMFRALYCLHKEGVVHRDIKPGNFLFSRKLSKGYLIDFNLAMDLKQK

[0179] HNIGSKSKPSHDAASNIVSFSSGSAPLVRDKNLGGSKSLTSNKRALADYKNYSELNRHVKQKDCTGPLKNCPDK

[0180] AGGSFLRAQGTDGSGVTSAKDPSTRTASAERLREPLPSHGRKELISFVNTMKCANNSSTIGPSSQRKRVTAPSS

[0181] KVDGKIFNITPMPLHSSTVGGGLMRSKGDGKKKEGSCVGTKGFRAPEVLLRSQHQGHKIDIWSAGVTLLYMVIG

[0182] KTPFTGDPEQNIKEIVKLRGSEEFWEVAKLHDRELSFPVELLDDRYLQSWDLEGWCKIHTKRPEFLEQIPKSLFDLIDKCLTVNPRNRLSAEDVLRHEFFDSLHESLRKQRMLHRHRALRSDAAASRAI。

[0183] The nucleic acid sequence (CDS) of the soybean GmCDC7 gene encoding sequence (SEQ ID NO.3, 2832bp) is sequence 3 in the sequence list, specifically as follows:

[0184]

[0185] The GmCDC7 gene knockout vector was constructed as follows: the upstream primer for the target sequence was primer-F: GGAT TGCCCTCCGTCAGCGCCGTCTTCGC-3', downstream primer is primer-R: 5'- AAAC GCGAAGACGGCGCTGACGGA CA-3' (The underlined base is used to form a sticky end).

[0186] Primers F and R were annealed to form annealed sgRNA (a double-stranded DNA fragment with sticky ends). pGES201 was digested with BsaI to obtain a linearized vector of pGES201. The sgRNA was ligated into the linearized vector of pGES201 to obtain the GmCDC7 gene knockout vector pCRISPR-Cas9-GmCDC7, which expresses Cas9 and sgRNA targeting the target sequence. pCRISPR-Cas9-GmCDC7 was introduced into competent cells of Agrobacterium tumefaciens EHA105 to obtain recombinant Agrobacterium EHA105 / pCRISPR-Cas9-GmCDC7. 1.2 Agrobacterium soybean genetic transformation

[0187] Soybean genetic transformation was performed by infecting soybean cotyledonary nodes with Agrobacterium tumefaciens containing the CRISPR-Cas9-GmCDC7 plasmid. The specific procedure included:

[0188] (1) Soybean seed cleaning and disinfection: The soybean variety Jack was used as the recipient material. Seeds with plump kernels, uniform size and no disease spots were selected. Sodium hypochlorite and concentrated hydrochloric acid were reacted to release chlorine gas for overnight disinfection.

[0189] (2) Explant preparation: Treated soybean seeds were placed in a clean Erlenmeyer flask and soaked overnight in sterile water. In a laminar flow hood, the seed coat was cut along the hilum using a scalpel, separating the embryo into two halves. The cotyledon explants were placed in a bacterial suspension with an OD value of 0.6-0.8 and ultrasonically treated for 3 minutes. The Erlenmeyer flask containing the explants was then placed in a vacuum pump and treated for 10 minutes (0.6 Pa). Finally, it was placed on a shaker and cultured at 23°C and 110 rpm for 40 minutes.

[0190] (3) Co-culture: After discarding the bacterial solution, spread the explants on clean filter paper, dry them, spread them on the co-culture medium, and place them in an incubator. After culturing in the dark at 23℃ for 3 days, transfer them to a tissue culture room at 23-25℃ with a photoperiod of 16h light / 8h darkness for 1 day.

[0191] (4) Recovery culture: Cut off part of the hypocotyl of the explant, leaving 3-4 mm, and insert it obliquely into the recovery culture medium and seal it. Place it in the tissue culture room with a photoperiod of 16h light / 8h darkness for 7 days.

[0192] (5) Bud induction and selection culture: Excessively long hypocotyls were removed, and the explants were inserted obliquely into the selection culture medium. They were placed in the tissue culture room with a photoperiod of 16h light / 8h darkness for 21 days.

[0193] (6) Bud elongation culture: Remove the cotyledons of the explant, retaining the clustered buds and base, and make a new wound at the base. Place on bud induction medium in a tissue culture room at 23-25℃ with a photoperiod of 16h light / 8h darkness for 2-8 weeks. During this period, change the medium or induce root growth according to the growth status of the explant.

[0194] (7) Root induction culture: When the shoots elongate to 3-5 cm, cut the elongated shoots from the base of the tissue, soak the cut in auxin solution for 2 minutes, and then transfer them into the rooting medium. Place them in the tissue culture room for root induction, which generally takes about 2 weeks.

[0195] (8) Seedling transplanting: Transplant the seedlings into the soil, cover them with a thin film and place them in the light. After the plants have grown normally for 3-5 days, remove the film to obtain T0 generation plants. Harvest T0 generation seeds, self-pollinate the T0 generation to obtain T1 generation seeds, and plant the T1 generation seeds to obtain T1 generation seedlings.

[0196] The co-culture medium formula is as follows: B5 medium + 3% sucrose + 0.8% Agar + 3mM MES + 0.25mg / L gibberellin (GA3) + 1.67mg / L benzylaminopurine (6-BA) + 580mg / L cysteine ​​+ 154.2mg / L dithiothreitol + 200μM acetoyl eugenol (AS), pH 5.4.

[0197] The recovery culture medium formula is: MS medium + 3% sucrose + 3mM MES + 0.8% Agar + 1.67mg / L benzylaminopurine (6-BA) + 100mg / L cephalosporin (Cef), pH 5.7.

[0198] The recovery culture medium formula is: MS medium + 3% sucrose + 3mM MES + 0.8% Agar + 1.67mg / L benzylaminopurine (6-BA) + 100mg / L cephalosporin (Cef), pH 5.7.

[0199] The screening medium formulation is as follows: MS medium + 3% sucrose + 3mM MES + 0.8% Agar + 1.67 mg / L benzylaminopurine (6-BA) + 5 mg / L Glufosinate + 100 mg / L cephalosporin (Cef), pH 5.7.

[0200] The bud induction medium formula is as follows: MS medium + 3% sucrose + 3mM MES + 0.8% Agar + 50mg / L asparagine + 50mg / L glutamine + 0.1mg / L indoleacetic acid (IAA) + 0.5mg / L gibberellin (GA3) + 1mg / L zeatin + 5mg / L glutfosinate + 500mg / L termethin (Tim) + 100mg / L cephalosporin (Cef), pH 5.7.

[0201] The rooting medium formula is: MS medium + 3% sucrose + 3mM MES + 0.8% Agar + 1mg / L IBA, pH 5.7.

[0202] 1.3 Genotypic and Phenotypic Identification of GmCDC7 Gene Mutant Plants Using the DNA of the obtained transgenic plants as templates, PCR amplification was performed on the plants using upstream primer F: 5'-CTCGATCCATGGCAGAGTC C-3' and downstream primer R: 5'-GGCTTCTGACTACCTGGAACG-3'. The PCR products were then subjected to Sanger sequencing to identify the mutation status of the target site in the T1 generation plants.

[0203] Using PCR and Sanger sequencing, four editing modes were identified in the target site mutations of GmCDC7-CRISPR-T1 generation plants (CR1-3bp, CR2-4bp, CR3-6bp, CR4-22bp).

[0204] The CR1-3bp and CR3-6bp edit types involve deletions of 3bp or 6bp at the target sites (bases 214 to 236 of SEQ ID No. 3; bases 422 to 444 of SEQ ID No. 1). These two mutation types result in amino acid deletions without causing frameshift mutations that prematurely terminate translation. However, the CR2-4bp and CR4-22bp edits result in deletions of 4bp or 22bp at the target sites, leading to frameshift mutations and premature termination of amino acid translation.

[0205] Specifically, in CR1 (CR1-3bp), compared with the soybean variety Jack, the region corresponding to the GmCDC7 gene in the genome underwent the following changes: 3 bp of TCA bases were deleted between positions 428 and 432 of the sequence shown in SEQ ID NO.1, resulting in the deletion of amino acids, but did not cause frameshift mutation to terminate translation prematurely.

[0206] In CR2 (CR2-4bp), compared with the soybean variety Jack, the region corresponding to the GmCDC7 gene in the genome has undergone the following changes: 4 bp of T CAG bases are deleted between positions 428 and 433 of the sequence shown in SEQ ID NO.1, resulting in a frameshift mutation and premature termination of amino acid translation.

[0207] In CR3 (CR3-6bp), compared with the soybean variety Jack, the region corresponding to the GmCDC7 gene in the genome has undergone the following changes: 6 bp of C CGTCA bases have been deleted between positions 425 and 432 of the sequence shown in SEQ ID NO.1, resulting in the deletion of amino acids, but without causing frameshift mutation to terminate translation prematurely.

[0208] In CR4 (CR4-22bp), compared with the soybean variety Jack, the region corresponding to the GmCDC7 gene in the genome has undergone the following changes: 22bp of base CGTCAGCGCCGTCTTCGCCCTC is deleted between positions 426 and 449 of the sequence shown in SEQ ID NO.1, resulting in a frameshift mutation and premature termination of amino acid translation.

[0209] Figure 1 This is a homozygous mutation of the GmCDC7 gene in T1 generation plants. Figure 1 A in the diagram represents the structure of the GmCDC7 gene and the location of the target Target1, specifically including the wild-type WT (soybean variety Jack) reference sequence and the CR1-3bp, CR2-4bp, CR3-6bp, and CR4-22bp mutant sequences; red represents the PAM sequence; short dashes represent base deletions. Figure 1 In Figure B, the sequencing peak diagrams of the wild-type and mutant sequences are shown, with the red areas indicating the locations where mutations occurred.

[0210] Example 2: Phenotypic identification of soybean seeds from GmCDC7 gene mutant plants

[0211] Seeds from mature mutant plants CR1, CR2, CR3, and CR4 were harvested and planted together with the wild-type soybean variety Jack in the summer of 2022 at the Molecular Breeding Experimental Base of the Institute of Botany, Chinese Academy of Sciences (116°20′E, 39°99′N). The plants were grown in pots using a substrate of potting soil and vermiculite in a 3:1 ratio. Ten pots were planted for each line, with two plants per pot. Phenotypic analysis was performed after maturity, with at least 10 biological replicates for each line.

[0212] After the potted soybean seeds matured, the seeds harvested from individual plants were dried at 37℃ for one week. Once the seeds reached constant weight, the grain traits of the wild-type soybean variety Jack and the gene mutant plants CR1, CR2, CR3, and CR4 were measured. Figure 2 Figure A shows the phenotypic diagram of the seeds of the GmCDC7 gene mutant plant and the wild type.

[0213] Seed length, width, and thickness statistics: Seed length, width, and thickness were also statistically analyzed for thoroughly dried seeds. Twenty seeds were taken from each line of the wild-type soybean variety Jack and the mutant plants CR1, CR2, CR3, and CR4. The biological experiment was repeated three times, and the results are expressed as mean ± standard deviation. A t-test was used, with P < 0.05 (*) indicating statistically significant differences, and P < 0.01 (**) and P < 0.001 (***) indicating highly significant differences. Figure 2 The results from Figure B indicate that, compared to the wild-type soybean variety Jack, the CR1-CR4 mutant plants have a significantly increased grain length of 5.55%-9.96%. Figure 2 The C-value indicates that the grain width of the mutant plant CR1 is significantly different from that of the wild-type soybean variety Jack. Figure 2 The results from the D study showed that, compared with the wild-type soybean variety Jack, the CR1-CR4 mutant plants had a significantly increased grain thickness of 2.71%-5.44%.

[0214] 100-seed weight statistics: The 100-seed weight of thoroughly dried seeds was counted. For the wild-type soybean variety Jack and the mutant plants CR1, CR2, CR3, and CR4, the 100-seed weight of at least three individual plants was measured. Each individual plant was randomly measured three times. The results are expressed as mean ± standard deviation. A t-test method was used. P < 0.05 (*) indicated a significant difference, and P < 0.01 (**) and P < 0.001 (***) indicated extremely significant differences. Figure 2 As can be seen from E, the weight of 100 seeds in the gene-mutated plants increased significantly by 13.07%-16.99%.

[0215] 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. A method for improving grain traits in a plant, comprising, The method includes improving the seed traits of a target plant by gene knockout, wherein the gene knockout is the knockout of the gene encoding the target protein in the target plant, and the target plant contains the gene encoding the target protein. The target protein is GmCDC7, and can be any of the following: A1) The amino acid sequence is that of the protein shown in sequence 2. A2) A protein obtained by substituting and / or deleting and / or adding amino acid residues of the protein described in A1), which has more than 80% identity with the protein shown in A1) and can regulate plant grain traits. A3) A fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of A1) or A2).

2. A method for cultivating plants with improved grain traits, comprising obtaining plants with improved grain traits by gene knockout, wherein the target plant contains the gene encoding the protein of claim 1.

3. The method according to claim 1 or 2, characterized in that, The protein is derived from soybeans.

4. The method as described in claims 1-3, characterized in that, The genome of the target plant contains a DNA molecule whose nucleotide sequence is sequence 1 in the sequence listing, and the gene knockout includes performing any of the following operations on the genome of the target plant: B1) Delete nucleotides 429-431 from sequence 1 in the sequence listing of the target plant. B2) Delete nucleotides 429-432 from sequence 1 in the sequence listing of the target plant. B3) Delete nucleotides 426-431 from sequence 1 in the sequence listing of the target plant. B4) Delete nucleotides 427-448 from sequence 1 in the sequence listing of the target plant.

5. The method according to any one of claims 1-4, characterized in that, The improved plant seed traits are selected from at least one of the following: C1) Increases the grain weight of plant seeds. C2) Increases the length of plant seeds. C3) Increases the thickness of plant seeds. C4) Increases the grain width of plant seeds.

6. The method according to any one of claims 1-5, characterized in that, The plant is any one of the following: J1) Dicotyledonous plants, J2) Plants of the Rosales order, J3) Leguminosae (family legumes) J4) Plants of the genus *Glycine*. J5) Soybeans.

7. The application of biomaterials, characterized in that, The application is any one of the following: D1) The application of the biomaterials described herein in improving plant seed traits and / or in preparing products that improve the seed traits of the target plant. D2) The application of the biomaterials described herein in plant breeding and / or the preparation of plant breeding products; The biological material is a protein, a substance that regulates the expression of the gene encoding the protein, or a substance that regulates the content of the protein. The protein is GmCDC7, and is any one of the following: The amino acid sequence of E1 is the protein shown in sequence 2. E2) A protein obtained by substituting and / or deleting and / or adding amino acid residues of the protein described in E1), which has more than 80% identity with the protein shown in E1) and can regulate plant grain traits. E3) is a fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of E1) or E2).

8. The application according to claim 7, characterized in that, The substance is any one of the following: F1) Nucleic acid molecules that inhibit, reduce, or downregulate the expression of the gene encoding the protein described in claim 7. F2) expresses the gene encoding the nucleic acid molecule described in F1). F3) contains the expression cassette of the gene described in F2). F4) a recombinant vector containing the gene described in F2), or a recombinant vector containing the expression cassette described in F3). F5) Recombinant microorganisms containing the gene described in F2), or recombinant microorganisms containing the expression cassette described in F3), or recombinant microorganisms containing the recombinant vector described in F4). F6) A transgenic plant cell line containing the gene described in F2), or a transgenic plant cell line containing the expression cassette described in F3), or a transgenic plant cell line containing the recombinant vector described in F4). F7) Transgenic plant tissue containing the gene described in F2), or transgenic plant tissue containing the expression cassette described in F3), or transgenic plant tissue containing the recombinant vector described in F4), F8) A transgenic plant organ containing the gene described in F2), or a transgenic plant organ containing the expression cassette described in F3), or a transgenic plant organ containing the recombinant vector described in F4).

9. The application as described in claim 7 or 8, characterized in that, The improved plant seed traits are selected from at least one of the following: H1) Increases the grain weight of plant seeds. H2) increases the length of plant seeds. H3) increases the thickness of plant seeds. H4) increases the grain width of plant seeds.

10. The application as described in any one of claims 7-9, characterized in that, The plant is any one of the following: J1) Dicotyledonous plants, J2) Rosales plants, J3) Leguminosae plants, J4) Glycine genus plants, J5) Soybean.

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