Application of protein GmMYB331 and related biological materials thereof in regulating and controlling grain weight character of seeds

Abstract

The invention belongs to the field of molecular biology, and particularly relates to application of protein GmMYB331 and related biological materials thereof in regulating and controlling the grain weight character of seeds. The GmMYB331 is a protein of which the amino acid sequence is as shown in SEQ ID No. 2. GmMYB331 overexpression strains OE1, OE2, OE3 and OE4 are obtained by introducing a gene for coding GmMYB331 into soybean Jack, and the grain weight of the GmMYB331 overexpression strains is obviously higher than that of a receptor control Jack, so that the GmMYB331 and the coding gene thereof can regulate the grain weight of plant seeds. The GmMYB331 and related biological materials thereof can be used for increasing the grain weight of seeds and cultivating high-yield varieties.

Description

Technical Field

[0001] The invention belongs to the field of molecular biology and relates to the application of protein GmMYB331 and related biological materials in regulating seed weight traits. Background Art

[0002] Soybeans are an important traditional crop, rich in nutritional value, and a crucial cash crop for providing food, oil, and feed. Soybean oil also has numerous industrial applications, such as biofuel, surfactants, and softeners. As the public's demand for healthy foods increases, soybean production is far from meeting this demand. Therefore, increasing soybean yield per unit area has become a pressing issue.

[0003] Seed weight (kernel weight) is an important indicator in crop production and one of the key agronomic traits affecting crop yield. Plants can increase yield by increasing seed weight.

[0004] Soybean yield is determined by factors such as plant architecture, pod-setting rate, pod number, and 100-grain weight. Grain weight is the most heritable factor. The impact of grain weight on yield is not limited to legumes; it also plays a crucial role in yield potential in other monocots and dicots, making it a crucial trait to consider during crop variety breeding. Previous studies have shown that grain weight is influenced by both the cultivation environment and genetics. Under normal cultivation conditions, genetics, specifically the relevant genes, play a significant role. Therefore, research on the molecular mechanisms underlying grain weight has become a hot topic. Summary of the Invention

[0005] The present invention aims to provide a soybean transcription factor, GmMYB331, and its encoding gene for use in regulating seed weight. The present invention is not limited to the technical subject matter described herein, and those skilled in the art will readily appreciate other technical subject matter not described herein through the following description.

[0006] In order to solve the above technical problems, the present invention provides the following technical solutions: The present invention provides the use of a protein or a substance that regulates the expression of a gene encoding the protein or a substance that regulates the activity or content of the protein. The protein is GmMYB331 protein, which can be any of the following: A1) a protein having an amino acid sequence of SEQ ID NO: 2, A2) a protein obtained by replacing and / or deleting and / or adding amino acid residues of the protein of A1) and having an identity of more than 80% with the protein of A1) and being related to regulating grain weight traits of seed plants, A3) A fusion protein obtained by linking the N-terminus or / and C-terminus of A1) or A2) to a protein tag; The application may be any of the following: M1) Regulates grain weight traits in seed plants; M2) Preparation of products for regulating grain weight traits in seed plants; M3) Cultivating seed plants with altered grain weight traits; M4) preparing products for cultivating plants with altered grain weight; M5) Plant breeding; M6) Preparation of products for use in plant breeding.

[0007] In the above application, the grain weight may be the weight of 100 grains.

[0008] In the above application, the change in grain weight may be an increase in grain weight.

[0009] In the above application, the protein may be derived from soybeans.

[0010] In the above-mentioned protein context, identity refers to amino acid sequence identity. Amino acid sequence identity can be determined using homology search sites on the Internet, such as the BLAST page on the NCBI homepage. For example, using Advanced BLAST 2.1, blastp can be used as the program, with the Expect value set to 10, all filters set to OFF, BLOSUM62 as the matrix, and the Gap existence cost, Per residue gap cost, and Lambda ratio set to 11, 1, and 0.85 (default values), respectively. The identity of a pair of amino acid sequences can be calculated and the identity value (%) can be obtained.

[0011] The aforementioned 80% or greater identity may be 80%, 85%, 90% or 95% or greater identity.

[0012] The 80% or greater identity may 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. The 85% or greater identity may be at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity. The 90% or greater identity may be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity. The 95% or greater identity may be at least 95%, 96%, 97%, 98%, or 99% identity.

[0013] The aforementioned SEQ ID NO: 2 consists of 331 amino acid residues.

[0014] The above proteins can be synthesized artificially, or their encoding genes can be synthesized first and then expressed biologically.

[0015] The present invention also provides applications of the biomaterial, which may be any of the following: C1) Use of the biological material in regulating the grain weight trait of seed plants and / or preparing products for regulating the grain weight trait of seed plants, C2) Use of the biological material in cultivating seed plants with altered grain weight traits and / or preparing products of cultivating seed plants with altered grain weight traits, C3) Use of the biological material in seed plant breeding and / or preparation of seed plant breeding products; The biological material is any one of the following: D1) a nucleic acid molecule encoding the protein; D2) an expression cassette containing the nucleic acid molecule 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) a recombinant microorganism containing the nucleic acid molecule described in D1), or a recombinant microorganism containing the expression cassette described in D2), or a recombinant microorganism 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 molecule described in D1), or transgenic plant tissue containing the expression cassette described in D2); D7) A transgenic plant organ containing the nucleic acid molecule described in D1), or a transgenic plant organ containing the expression cassette described in D2).

[0016] In the above-mentioned biological materials, the nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA.

[0017] Furthermore, in the above biological material, the nucleic acid molecule encoding the GmMYB331 in D1) can be GmMYB331 The GmMYB331 gene encodes GmMYB331 The coding sequence may be a DNA molecule having SEQ ID NO: 1 (SEQ ID NO: 1 in the sequence listing), wherein SEQ ID NO: 1 consists of 996 nucleotides, and its coding sequence is nucleotides 1 to 993 of SEQ ID NO: 1, encoding the protein shown in SEQ ID NO: 2.

[0018] Furthermore, in the above biological materials, the expression cassette described in D2) refers to a DNA encoding GmMYB331 that can express in a host cell, and the DNA may include not only a promoter GmMYB331 The promoter of gene transcription may also include the terminator GmMYB331The expression cassette may further comprise an enhancer sequence. Promoters useful in the present invention include, but are not limited to, constitutive promoters, tissue-, organ-, and development-specific promoters, and inducible promoters. Examples of promoters include, but are not limited to, the constitutive promoter 35S of cauliflower mosaic virus; a wound-inducible promoter from tomato, leucine aminopeptidase ("LAP," Chao et al. (1999) Plant Physiol 120:979-992); a chemically inducible promoter from tobacco, pathogenesis-related 1 (PR1) (induced by salicylic acid and BTH (benzothiadiazole-7-thiocarboxylic acid S-methyl ester)); the tomato proteinase inhibitor II promoter (PIN2) or the LAP promoter (both inducible by methyl jasmonate); a heat shock promoter ( U.S. Pat. No. 5,187,267 ); a tetracycline-inducible promoter ( U.S. Pat. No. 5,057,422 ); a seed-specific promoter, such as the millet seed-specific promoter pF128 ( CN101063139B ( China Patent No. 2007 1 0099169.7)), seed storage protein-specific promoters (e.g., the promoters of phaseolin, napin, oleosin, and soybean beta-conglycin (Beachy et al. (1985) EMBO J. 4:3047-3053)). These can be used alone or in combination with other plant promoters. All references cited herein are incorporated by reference in their entirety. Suitable transcription terminators include, but are not limited to, the Agrobacterium nopaline synthase terminator (NOS terminator), the cauliflower mosaic virus CaMV 35S terminator, the tml terminator, the pea rbcS E9 terminator, and the nopaline and octopine synthase terminators (see, e.g., Odell et al. (1985) Nature 313:810; Rosenberg et al. (1987) Gene, 56:125; Guerineau et al. (1991) Mol. Gen. Genet, 262:141; Proudfoot (1991) Cell, 64:671; Sanfacon et al. Genes Dev., 5:141; Mogen et al. (1990) Plant Cell, 2:1261; Munroe et al. (1990) Gene, 91:151; Ballad et al. (1989) Nucleic Acids Res. 17:7891; Joshi et al. (1987) Nucleic Acid Res., 15:9627).

[0019] Furthermore, in the biological material, D3) the recombinant vector may be a gene encoding the protein GmMYB331 GmMYB331 The recombinant expression vector of the expression cassette. The plant expression vector can be a Gateway system vector or a binary Agrobacterium vector, such as pGWB411, pGWB412, pGWB405, DTS9005, pBin438, pBI121, pCAMBIA1302, pCAMBIA2300, pCAMBIA1301, pCAMBIA1300, pCAMBIA1391-Xa or pCAMBIA1391-Xb. GmMYB331 When constructing a recombinant expression vector, any enhancing, constitutive, tissue-specific, or inducible promoter, such as the cauliflower mosaic virus (CAMV) 35S promoter or the ubiqutin gene promoter (pUbi), can be added before the transcription initiation nucleotide. These promoters can be used alone or in combination with other plant promoters. Furthermore, when constructing a plant expression vector using the gene of the present invention, enhancers, including translation enhancers and transcription enhancers, can be used. These enhancer regions can be the ATG start codon or the start codon in adjacent regions, but must be in frame with the coding sequence to ensure correct translation of the entire sequence. The translation control signals and start codons can be derived from a variety of sources, including natural or synthetic sources. The translation initiation region can be derived from the transcription initiation region or a structural gene.

[0020] In order to facilitate the identification and screening of transgenic plant cells or plants, the plant expression vector used can be processed, such as adding genes that can be expressed in plants and encode enzymes or luminescent compounds that can produce color changes (GUS gene, luciferase gene, etc.), antibiotic resistance markers (gentamicin marker, kanamycin marker, etc.) or chemical resistance marker genes (such as herbicide resistance genes), etc.

[0021] Furthermore, in the biological material, the recombinant microorganism D4) can specifically be yeast, bacteria, algae and fungi.

[0022] Furthermore, the recombinant microorganism may be Agrobacterium, and the Agrobacterium is LBA4404.

[0023] Furthermore, in the biological material, the plant tissue in D6) may be derived from roots, stems, leaves, flowers, fruits, seeds, pollen, embryos and / or anthers.

[0024] Furthermore, in the biological material, the transgenic plant organ in D7) can be a root, stem, leaf, flower, fruit and seed of a transgenic plant.

[0025] The plant breeding index includes the seed plant grain weight trait. The plant breeding purpose includes improving the plant grain weight trait.

[0026] The present invention also provides a method for regulating the grain weight trait of seed plants, which comprises the step of regulating the grain weight trait of seed plants by regulating the expression of the coding gene containing the above-mentioned protein in the target seed plants.

[0027] The present invention also provides a method for producing seed plants with altered grain weight traits, which includes the steps of regulating the expression of the coding gene containing the above-mentioned protein in the target seed plant to obtain the seed plant with altered grain weight traits.

[0028] In the above method, the expression of the coding gene in the target seed plant containing the coding gene of the protein can be specifically regulated by introducing the coding gene of GmMYB331 into the recipient seed plant by carrying the protein of the present invention. GmMYB331 The plant expression vector of the present invention is introduced into the recipient seed plant. GmMYB331 Plant expression vectors can be transformed into plant cells or tissues by conventional biological methods such as Ti plasmids, Ri plasmids, plant virus vectors, direct DNA transformation, microinjection, electroporation, Agrobacterium-mediated, etc., and the transformed plant cells or tissues can be cultivated into plants.

[0029] Carrying the present invention GmMYB331 The plant expression vector may be DTS9005- GmMYB331 .DTS9005 - GmMYB331 It is obtained by recombining the DNA molecule shown in sequence 1 in the sequence list into the vector DTS9005 GmMYB331 Gene expression vector.

[0030] In the above, the regulation can be at least one of the following 6 types of regulation: E1) regulation at the transcriptional level of the coding gene, E2) is a post-transcriptional regulation of the coding gene. E3) regulation of the transport of RNA encoding the gene, E4) regulation of the translation of the coding gene, E5) regulation of the degradation of the mRNA encoding the gene, E6) Post-translational regulation of the genes.

[0031] The modulation may be upregulation or enhancement or increase.

[0032] The above-mentioned protein can be derived from soybeans.

[0033] In the above, the seed plant may be any one of the following: F1) dicotyledons, F2) Rosales, F3) Leguminosae, F4) Glycine max, and F5) soybean.

[0034] The present invention also protects a product, which is the above-mentioned protein or biological material.

[0035] The present invention has been experimentally proved that GmMYB331 The grain weight of the transgenic soybeans introduced into soybean Jack was significantly higher than that of the recipient control Jack. GmMYB331 The 100-grain weights of the overexpression lines OE1, OE2, OE3, and OE4 were approximately 15.8, 23.3, 18.2, 17.3, and 16.3 g, respectively. GmMYB331 The 100-grain weights of the seeds in these strains were 47.5%, 15.2%, 9.5%, and 4.94% higher than those in the control, respectively, indicating that GmMYB331 and its encoding gene positively regulate plant seed weight and that overexpression increases plant seed weight. GmMYB331 Its related biological materials can be used to increase the 100-grain weight of seeds and cultivate high-yield varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 Plant expression vectors DTS9005 and DTS9005- GmMYB331 Schematic diagram of the structure.

[0037] Figure 2 for GmMYB331 Molecular testing of overexpression lines.

[0038] Figure 3 for GmMYB331 Comparison of seed phenotype and 100-grain weight between the transgenic line and the control Jack. A is the comparison of seed shape between the control and transgenic line, and B is the statistics of 100-grain weight between the control and transgenic line. DETAILED DESCRIPTION

[0039] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0040] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0041] The methods used in the following examples are conventional methods unless otherwise specified, and the primers used are synthesized by Sanbo Biotechnology Co., Ltd.

[0042] Soybean material: Soybean variety Jack is recorded in the document “Jin-Song Zhang1, et al., Atranscriptional regulatory module controls lipid accumulation in soybean, New Phytologist (2021), 231:661-678”.

[0043] Heinong 44 (HN44) was bred by the Soybean Research Institute of Heilongjiang Academy of Agricultural Sciences in 1998 through sexual hybridization of Heinong 37 as the female parent and Jilin 20 as the male parent using the pedigree method. The original variety number was Ha 94-4478. In 2002, it was approved and promoted by the Heilongjiang Provincial Crop Variety Approval Committee and named Heinong 44.

[0044] Soybean ZYD7 is described in the following literature: Xiang Lu et al., A PP2C-1 Allele Underlying a Quantitative Trait Locus Enhances Soybean 100-Seed Weight, Molecular Plant, Volume 10, Issue 5, 1 May 2017, Pages 670-684.

[0045] Agrobacterium LBA4404: purchased from Invitrgen, CAT: 18313-015, described in the non-patent literature "Joyce VanEck et al., Agrobacterium tumefaciens-Mediated Transformation of Tomato, Methods Mol Biol. 2019: 1864: 225-234. doi: 10.1007 / 978-1-4939-8778-8_16."

[0046] The above materials are available to the public from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences. The biological materials are only used to repeat the relevant experiments of the present invention and cannot be used for other purposes.

[0047] The data in the following examples were processed using GraphPad Prism 8 statistical software. The experimental results were expressed as mean ± standard deviation and tested using One-way ANOVA. P < 0.05 (*) indicated a significant difference.

[0048] Example 1 GmMYB331 Obtaining gene overexpression plants 1.1 Soybean transcription factor GmMYB331 Screening of coding genes and their cDNA cloning Using soybean varieties Heinong 44 (HN44) and wild soybean ZYD7 as materials, transcriptome analysis during seed development was conducted to construct a transcription factor co-regulatory network. Differentially expressed genes during seed development between Heinong 44 (HN44) and ZYD7 were obtained, from which candidate genes related to seed weight were further screened. The candidate genes were transformed into the model plant Arabidopsis thaliana, and the new version of the Williams82 reference genome was obtained. Glyma.20G184500 The gene may be related to seed weight. Since the gene is not named in the database, it is temporarily named GmMYB331 .

[0049] Total RNA was extracted from Hei Nong 44 seedlings, and the RNA was reverse transcribed using reverse transcriptase to synthesize cDNA (HN44 cDNA).

[0050] According to the soybean genome sequence in PlantGDB GmMYB331 ( Glyma.20G184500 ) The full-length cDNA sequence information was used to design primers. The primer sequences are as follows: Glyma.20G184500-F: 5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGTCTGACCCTTCTACCCTCTTAC-3'; Glyma.20G184500-R: 5'-GGGGACCACTTTGTACAAGAAAGCTGGGTTTTTATCTTTGGCCATGGCGTTCAG-3'.

[0051] Using the cDNA of HN44 as a template and the above primers, PCR amplification was performed to obtain a PCR product of about 1.0 Kb. After sequencing, the PCR product was 996 bp and had the nucleotide sequence shown in sequence 1 in the sequence table. The gene indicated by the nucleotide sequence is GmMYB331 The protein encoded by this gene is named GmMYB331, which contains 331 amino acids and its amino acid sequence is sequence 2 in the sequence table. GmMYB331 The sequence is sequence 1 in the sequence listing.

[0052] Sequence 2 (SEQ ID No. 2) contains 331 amino acids, as follows: MSDPSTLLPSSHHHHNHHVPLIQGGATAPSSSSTTLAREYRKGNWTIQETLILITAKKLDDERRLKTPAACSTSTTTTRTSGELRWKWVENYCWSHGCLRSQNQCNDKWDNLLRDYKKVRDYESKSNDNDNNNNKHFPSYWTLNKQQRKEQNLPSNMVFEVYQTIADVLQRKQTQSQRQHQQPLAIPLVTSSPSPLQTLPPPPLPPPPPPPPPPPPVSSTTPVGSERSESSGTEHSEDDDDGSESKRRKVKNLGSRIMQSASVLARALRSCEEKKEKRHREMIELEQRRIQMEEARNEVHRQGIATLVAAVTNLSGAIESLINNSERHGQR。

[0053] Sequence 1 (SEQ ID No.1) contains 996 bp and is as follows:

[0054] 1.2 Construction of plant expression vector and acquisition of recombinant Agrobacterium GmMYB331 Overexpression transgenic soybeans were obtained by Agrobacterium-mediated transformation. The Agrobacterium-mediated transformation method used recombinant Agrobacterium to express GmMYB331 The recombinant vector DTS9005- GmMYB331 The recombinant vector DTS9005- was obtained by electroporation into Agrobacterium tumefaciens LBA4404 (Invitrgen, Cat: 18313-015). GmMYB331 The plant expression vector DTS9005 (Beijing Dabeinong Biotechnology Co., Ltd., Figure 1 Middle A) The vector DTS9004 was double-digested with SalI and SpeI, and the digestion product was purified. The cDNA was amplified from the cDNA of HN44 using primers 9004-P1-F: 5'-ttggagaggacagtcgacatgtctgacccttctaccctcttacc-3' and 9004-P1-R: 5'-cagatctgagtccggatctttggccatggcgttcagaatt-3'. GmMYB331 The CDS sequence (without the stop codon) was amplified and purified; the 4×MYC tag sequence was amplified and purified using primers 9004-P2-F: 5'-ggccaaagatccggactcagatctgagc-3', 9004-P2-R: 5'-gtttgaacgatcactagtttatcacaagtcctcttcagaaatgagc-3'. Takara's In-Fusion seamless ligation product kit (Clontech, Catalog No: 639649) was used to amplify the 4×MYC tag sequence. GmMYB331 The gene fragment, 4×MYC (MYB) tag fragment and linearized DTS9004 vector were homologously recombined. The recombinant vector was transformed into E. coli T1 competent cells using the heat shock method, and the plasmid was extracted for sequencing verification. The recombinant plasmid verified to be correct by sequencing was named DTS9005- GmMYB331 .DTS9005- GmMYB331 The DNA fragment containing the nucleotide sequence of SEQ ID No. 3, wherein the 1st to 993rd position of the GmMYB331 gene ( GmMYB331 The CDS of the gene is deleting the stop codon sequence), positions 994-1008 are the linker sequence, positions 1009-1128 are the 4×MYC (MYB) tag sequence, and positions 1129-1134 are TGATAA. GmMYB331 Can be expressed in soybean GmMYB331 .

[0055] Sequence 3 (SEQ ID No. 3, 1134 bp) in the sequence listing is as follows:

[0056] 1.3 Overexpression GmMYB331 Acquisition and identification of soybean Recombinant Agrobacterium LBA4404 / GmMYB331 After culturing to the logarithmic phase, the plants were transformed into soybean Jack using the cotyledonary node transformation method to obtain T0 generation plants. After incubation, the seeds were harvested. The seeds were sown in vermiculite and maintained in an ambient humidity of 70-75%, with a photoperiod of 16 hours at 11,000 Lux and 8 hours in darkness, and a temperature of 30-37°C during the day and 25-28°C at night. Soybean leaves were smeared with 0.1% Roundup (glyphosate). Plants showing no yellowing after three days were considered transgenic plants. A total of eight transgenic plants were obtained.

[0057] The above positive plants were subjected to molecular detection. RNA of transgenic plant seedlings was extracted and reverse transcribed to obtain cDNA as a template. The primers were: GmMYB331PCR-1F: 5'-CCTCACCCTCACCACTTCAAAC-3' and GmMYB331PCR-1R: 5'-CGATCTCTCTGAGCCTACCGG-3', and Real Time-PCR identification was performed. The soybean GmTubulin gene was used as an internal standard, and the primers used were Primer-TF and Primer-TR. Primer-TF: 5'-AACTCCATTTCGTCCATTCCTTC-3', and Primer-TR: 5'-TTGAGTGGATTCCCAACAACG-3'. The recipient Jack and the empty vector strain were used as controls. The biological experiment was repeated three times, and the results were taken as the mean ± standard deviation. GmMYB331 Four strains with different expression levels, OE1, OE2, OE3 and OE4, were further phenotypic analyzed. Figure 2 As shown, the transgenic lines OE1, OE2, OE3 and OE4 GmMYB331 The relative expression levels of α, β, β and β were approximately 2.4±0.90, 1.8±0.41, 5.6±0.59 and 4.1±0.95, respectively. GmMYB331 The relative expression level was very low, approximately 0.0016±0.0008. The four transgenic lines were propagated to the T3 generation. Each individual plant in each generation had its leaves smeared with 0.1% Roundup (glyphosate). Plants that showed no yellowing after three days were considered transgenic-positive and tested. Negative plants were eliminated, resulting in pure transgenic lines with no segregation in the offspring.

[0058] The above results further prove that GmMYB331 The four strains OE1, OE2, OE3 and OE4 are GmMYB331 Overexpression lines.

[0059] Example 2: Overexpression GmMYB331 Increased soybean seed weight 2.1 Overexpression GmMYB331 Phenotype of strains Test soybean: overexpression in Example 1 GmMYB331 Lines OE1, OE2, OE3 and OE4 and the wild-type soybean Jack control.

[0060] Soybean greenhouse pot cultivation conditions: Sow soybean seeds in pots filled with vermiculite. Maintain humidity at 70-75% during growth. Greenhouse conditions: 16 hours of light at 11,000 Lux and 8 hours of darkness, with temperatures ranging from 30 to 37°C during the day and 25 to 28°C at night. Observe the growth and development of the soybeans. Harvest the seeds after 130 days. No significant differences were observed during the soybean growth period.

[0061] 2.2 Overexpression GmMYB331 100-grain weight test of strains The control Jack and genetically stable transgenic lines OE1, OE2, OE3 and OE4 were planted in greenhouse pots under the above-mentioned conditions and harvested after 130 days. After the seeds matured, the 100-grain weight of the recipient Jack, transgenic lines OE1, OE2, OE3 and OE4 seeds was measured. The mature seeds harvested from each plant were then cultured for 37 days. o After drying for one week, five plants from each strain were selected, and all seeds were collected from each plant. The total weight of the thoroughly dried seeds was measured. The biological experiment was repeated three times, and the results are presented as mean ± standard deviation. One-way ANOVA was used to test the difference. P < 0.05 (*) indicates a significant difference, and P < 0.01 (**) indicates a very significant difference.

[0062] The results are as follows Figure 3 As shown in middle A, the seeds of transgenic lines OE1, OE2, OE3, and OE4 were significantly larger than those of the recipient control, Jack. The 100-grain weights of Jack and OE1, OE2, OE3, and OE4 seeds were 15.9±0.81, 23.3±0.75, 18.2±0.78, 17.3±1.2, and 16.3±1.3 g, respectively. GmMYB331 The 100-grain weights of OE1, OE2, OE3, and OE4 were 47.5%, 15.2%, 9.5%, and 4.94% higher than those of the control, respectively. The 100-grain weights of OE1 and OE2 were extremely significantly higher than those of the control, while that of OE3 was significantly higher than that of the control.

[0063] The above results indicate that GmMYB331 positively regulates soybean seed weight and upregulates / increases the GmMYB331 encoding gene GmMYB331 The expression level of β-catenin can significantly increase soybean seed weight.

[0064] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need for unnecessary experimentation, the present invention can be implemented in a wider range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In short, according to the principles of the present invention, this application is intended to include any changes, uses or improvements to the present invention, including changes that depart from the scope disclosed in this application and are made with conventional techniques known in the art.

Claims

1. Use of a protein or a substance that regulates the expression of a gene encoding the protein or a substance that regulates the activity or content of the protein, characterized in that: The protein is GmMYB331 protein, which is any of the following: A1) a protein having an amino acid sequence of SEQ ID NO: 2, A2) a protein obtained by replacing and / or deleting and / or adding amino acid residues of the protein of A1) and having more than 80% identity with the protein shown in A1) and being related to regulating the grain weight trait of seed plants, A3) A fusion protein obtained by connecting the N-terminus or / and C-terminus of A1) or A2) to a protein tag; The application is any of the following: M1) regulates the grain weight traits of seed plants, M2) Preparation of products for regulating grain weight traits of seed plants, M3) Cultivate seed plants with altered grain weight traits, M4) preparing products for breeding seed plants with altered grain weight traits, M5) Seed plant breeding, M6) Preparation of products for seed plant breeding.

2. The use according to claim 1, characterized in that: The protein is derived from soy beans.

3. The use according to claim 1 or 2, characterized in that: The regulation is at least one of the following 6 types of regulation: B1) Regulation at the transcriptional level of the coding gene, B2) regulation after transcription of the coding gene, B3) regulation of the transport of RNA encoding the gene, B4) regulation of the translation of the coding gene, B5) regulation of the degradation of the mRNA encoding the gene, B6) Post-translational regulation of the gene.

4. Application of biomaterials, characterized in that The application is any of the following: C1) Use of the biological material in regulating the grain weight trait of seed plants and / or preparing products for regulating the grain weight trait of seed plants, C2) Use of the biological material in cultivating seed plants with altered grain weight traits and / or preparing products of cultivating seed plants with altered grain weight traits, C3) Use of the biological material in seed plant breeding and / or preparation of seed plant breeding products; The biological material is any of the following: D1) a nucleic acid molecule encoding the protein according to claim 1 or 2, D2) an expression cassette containing the nucleic acid molecule 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) a recombinant microorganism containing the nucleic acid molecule described in D1), or a recombinant microorganism containing the expression cassette described in D2), or a recombinant microorganism 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 molecule described in D1), or transgenic plant tissue containing the expression cassette described in D2), D7) A transgenic plant organ containing the nucleic acid molecule described in D1) or a transgenic plant organ containing the expression cassette described in D2).

5. A method for regulating the grain weight trait of seed plants, characterized in that: The method comprises the step of regulating the grain weight trait of seed plants by regulating the expression of the coding gene of the protein in the target seed plants containing the coding gene of the protein in claim 1 or 2.

6. A method for producing seed plants with altered grain weight traits, characterized in that: The method comprises the steps of obtaining a seed plant with altered grain weight trait by regulating the expression of the coding gene of the protein in the target seed plant containing the coding gene of the protein in claim 1 or 2.

7. The method according to claim 5 or 6, characterized in that: The regulation is at least one of the following 6 types of regulation: E1) regulation at the transcriptional level of the coding gene, E2) is a post-transcriptional regulation of the coding gene. E3) regulation of the transport of RNA encoding the gene, E4) regulation of the translation of the coding gene, E5) regulation of the degradation of the mRNA encoding the gene, E6) Post-translational regulation of the gene.

8. The method according to claim 5, 6 or 7, characterized in that: The protein is derived from soy beans.

9. The use according to claims 1 to 4 or the method according to claims 5 to 8, characterized in that: The seed plant is any one of the following: F1) Dicotyledons, F2) Plants of the order Rosales, F3) Leguminosae, F4) soybean plants, F5) Soybean.

10. A product, characterized in that The product is the protein described in claim 1 or the biological material described in claim 4.

Images