Application of soybean protein GmSFR2 related to seed weight regulation and its related biomaterials

Abstract

The present invention discloses the application of soybean protein GmSFR2 related to seed weight regulation and related biomaterials, and the technical problem to be solved is how to increase the weight of plant seeds. The present application discloses any of the following applications of GmSFR2 protein, a substance that upregulates, enhances or increases the expression of the gene encoding the protein, or a substance that upregulates, enhances or increases the content of the protein: A1) increasing plant seed weight; A2) preparing products for increasing plant seed weight; A3) cultivating high-grain weight seed plants; A4) preparing products for cultivating high-grain weight seed plants. The seed weight of the overexpressing plant that overexpresses the gene encoding the GmSFR2 protein is significantly higher than that of the wild-type recipient plant. The substance that upregulates, enhances or increases the expression of the GmSFR2 gene can be used to increase plant seed weight or for plant high seed weight breeding.

Description

Technical Field

[0001] The present invention specifically relates to application of soybean protein GmSFR2 related to seed weight regulation and related biomaterials. Background Art

[0002] Soybean is an important traditional crop with rich nutritional value. It is an important economic crop that provides food, oil and feed. Soybean oil also has many uses in industrial production, such as biofuel, surfactant, softener, etc.

[0003] Seed weight (grain weight) is an important indicator in crop production and one of the important agronomic traits that affect crop yield. Plants can achieve the goal of increasing yield by increasing grain weight.

[0004] Soybean yield is composed of factors such as plant type, pod setting rate, number of pods, and seed weight of 100 seeds, among which grain weight is the factor with the highest heritability. The effect of grain weight on yield is not limited to legumes. It is also an important factor in yield potential for other monocotyledonous and dicotyledonous plants. Therefore, it has become an important selective trait to be considered in the process of crop variety breeding. Existing studies have shown that grain weight is affected by cultivation environment and genetics. Under normal cultivation conditions, genetics, that is, related genes, play an important role. Therefore, the study of molecular mechanisms related to grain weight has become a hot topic.

[0005] Glycoside hydrolase, also known as glycosidase, is a large class in the hydrolase family. It belongs to the glycosylase and is a general term for enzymes that act on various glycosides or oligosaccharides to hydrolyze glycosidic bonds. The enzymatic number is EC 3.2.1. In the glycoside hydrolase family, according to the different substrates hydrolyzed, it can be divided into 195 different types of enzymes. It is known that there are many types of glycoside hydrolases in organisms, which play an important role in plant growth, development, stress tolerance, etc. For example, the glycoside hydrolase 32 family (GH32) is responsible for the hydrolysis or synthesis of fructan glycosidic bonds, and plays a role in cell wall metabolism, cell differentiation and development, plant defense, etc.; GH7 acts on the β-1,4-bonds in the crystalline or amorphous regions of cellulose, which can be used to efficiently degrade cellulose into fermentable sugars; GH61 combined with cellulase can decompose cellulose into fermentable sugars, etc. The present application locates the QTL related to grain weight through the recombinant inbred line Kefeng 1 (small grain weight) / Nannong 1138-1 (large grain weight), and identifies the gene Glyma.17G037100 encoding the glycoside hydrolase superfamily protein in the QTL interval located on chromosome 17. After investigation, the gene responds to freezing and is related to freezing tolerance, so it is named GmSFR2. There are no reports on the protein and such enzymes and the regulation of seed weight. Summary of the invention

[0006] The invention discloses soybean protein GmSFR2 related to seed weight regulation and application of related biomaterials.

[0007] The technical problem to be solved by the present invention is how to increase the weight of plant seeds, particularly Arabidopsis thaliana.

[0008] In order to solve the above problems, the present application provides the following application:

[0009] The protein, the substance that up-regulates, enhances or increases the expression of the gene encoding the protein, or the substance that up-regulates, enhances or increases the activity or content of the protein is used in any of the following:

[0010] A1) Increase plant seed weight;

[0011] A2) preparing a product for increasing the weight of plant seeds;

[0012] A3) Cultivating high seed weight plants;

[0013] A4) Preparation and cultivation of high seed weight plant products.

[0014] The protein is any one of the following:

[0015] B1) The amino acid sequence is the protein shown in SEQ ID NO: 2;

[0016] B2) a protein obtained by substitution and / or deletion and / or addition of amino acid residues of the protein described in B1) which has more than 80% identity with the protein described in B1) and has the function of up-regulating, enhancing or increasing plant seed weight;

[0017] B3) A fusion protein obtained by connecting the N-terminus and / or C-terminus of B1) or B2) to a protein tag.

[0018] In the above proteins, the protein tag refers to a polypeptide or protein that is fused and expressed with the target protein using DNA in vitro recombination technology to facilitate the expression, detection, tracing and / or purification of the target protein. The protein tag can be a Flag tag, a His tag, an MBP tag, an HA tag, a myc tag, a GST tag and / or a SUMO tag, etc.

[0019] In the above-mentioned proteins, identity refers to the identity of the amino acid sequence. The identity of the amino acid sequence can be determined using a homology search site on the Internet, such as the BLAST page on the NCBI homepage website. For example, in Advanced BLAST2.1, by using blastp as a program, setting the Expect value to 10, setting all Filters to OFF, using BLOSUM62 as a Matrix, setting the Gap existence cost, Per residue gap cost and Lambda ratio to 11, 1 and 0.85 (default values) respectively, and searching for the identity of a pair of amino acid sequences, the identity value (%) can be obtained.

[0020] In the above proteins, the above 80% identity may be at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 95%, 96%, 98%, 99% or 100% identity.

[0021] In the above protein, sequence 2 (SEQ ID No. 2) consists of 637 amino acid residues

[0022] In the above, the substance that regulates gene expression may be a substance that performs at least one of the following six types of regulation: 1) regulation at the transcription level of the gene; 2) regulation after transcription of the gene (that is, regulation of the splicing or processing of the primary transcript of the gene); 3) regulation of RNA transport of the gene (that is, regulation of the transport of the mRNA of the gene from the nucleus to the cytoplasm); 4) regulation of the translation of the gene; 5) regulation of the degradation of the mRNA of the gene; 6) post-translational regulation of the gene (that is, regulation of the activity of the protein translated from the gene).

[0023] In the above application, the protein is derived from soybeans.

[0024] The protein may be named GmSFR2, and may be derived from soybeans. The soybeans may be Nannong 1138-2.

[0025] Herein, the substance that regulates the activity and / or content of the protein may be a substance that regulates gene expression, and the protein encoded by the gene is any of the following:

[0026] C1) The amino acid sequence is the protein shown in SEQ ID NO: 2;

[0027] C2) A protein obtained by replacing and / or deleting and / or adding amino acid residues of the protein described in C1) and having an identity of more than 80% with the protein described in C1) and having the ability to increase plant seed weight.

[0028] In order to solve the above problems, the present application also provides the application of relevant biological materials.

[0029] Application of relevant biomaterials in any of the following;

[0030] The biological material is any of the following:

[0031] D1), a nucleic acid molecule encoding the protein according to claim 1 or 2;

[0032] D2), an expression cassette containing the nucleic acid molecule described in D1);

[0033] D3), ​​a recombinant vector containing the nucleic acid molecule described in D1), or a recombinant vector containing the expression cassette described in D2);

[0034] 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);

[0035] 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), or a transgenic plant cell line containing the recombinant vector described in D3);

[0036] D6), transgenic plant tissue containing the nucleic acid molecule described in D1), or transgenic plant tissue containing the expression cassette described in D2), or transgenic plant tissue containing the recombinant vector described in D3);

[0037] 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), or a transgenic plant organ containing the recombinant vector described in D3);

[0038] The application is any of the following:

[0039] A1) Increase plant seed weight;

[0040] A2) preparing a product for increasing the weight of plant seeds;

[0041] A3) Cultivating high seed weight plants;

[0042] A4) Preparation and cultivation of high-grain weight seed plant products. In the above application, the gene encoding the protein is any one of the following:

[0043] E1) The nucleic acid sequence is the DNA shown in Sequence 1;

[0044] E2) A DNA having an identity of 80% or more to the DNA shown in E1) and having the ability to increase plant seed weight obtained by substituting and / or deleting and / or adding nucleotide residues to the DNA shown in E1).

[0045] The substance that upregulates, enhances or improves the expression of the gene encoding the protein can be the above-mentioned D1) nucleic acid molecule encoding the above-mentioned protein, D2) an expression cassette containing the nucleic acid molecule D1) or D3) a recombinant vector containing the nucleic acid molecule D1) or a recombinant vector containing the expression cassette D2).

[0046] B1) In the nucleic acid molecule, a person skilled in the art can easily mutate the nucleotide sequence encoding the protein GmSFR2 of the present invention by using known methods, such as directed evolution or point mutation. Those artificially modified nucleotides having 80% or more identity with the nucleotide sequence of the protein GmSFR2 isolated from the present invention are all derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention as long as they encode the protein GmSFR2 and have the function of the protein GmSFR2.

[0047] The above 80% or more identity may be 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

[0048] Herein, identity refers to the identity of an amino acid sequence or a nucleotide sequence. The identity of an amino acid sequence can be determined using a homology search site on the Internet, such as the BLAST webpage on the NCBI homepage website. For example, in Advanced BLAST2.1, by using blastp as a program, setting the Expect value to 10, setting all Filters to OFF, using BLOSUM62 as a Matrix, setting Gap existence cost, Per residue gap cost and Lambda ratio to 11, 1 and 0.85 (default values) respectively and searching to calculate the identity of an amino acid sequence, the value (%) of identity can then be obtained.

[0049] In the above biological material, the nucleic acid molecule in B1) may be a gene encoding the protein. Specifically, the nucleic acid molecule in B1) may be a DNA molecule whose coding sequence of the coding chain is shown in Sequence 1.

[0050] Herein, the vector is well known to those skilled in the art, including but not limited to: plasmid, phage (such as lambda phage or M13 filamentous phage, etc.), cosmid (i.e., cosmid), Ti plasmid or viral vector. Specifically, it can be vector pCR8 / GW / TOPO and / or pGWB411;

[0051] In the above biological materials, the expression cassette described in B2) refers to a DNA capable of expressing the gene in a host cell, and the DNA may include not only a promoter for initiating gene transcription, but also a terminator for terminating gene transcription. Further, the expression cassette may also include an enhancer sequence. Promoters that can be used 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); chemically inducible promoter from tobacco, pathogenesis-related 1 (PR1) (induced by salicylic acid and BTH (benzothiadiazole-7-thiocarboxylic acid S-methyl ester)); tomato proteinase inhibitor II promoter (PIN2) or LAP promoter (both can be induced by methyl jasmonate); heat shock promoter (U.S. Pat. No. 5,187,267); tetracycline-inducible promoter (U.S. Pat. No. 5,057,422); seed-specific promoters, such as millet seed-specific promoter pF128 (CN101063139B (Chinese Patent 200710099169.7)), seed storage protein-specific promoters (e.g., promoters of phaseolin, napin, oleosin and soybean beta conglycin (Beachy et al. (1985) EMBO J. 4:3047-3053)). They can be used alone or in combination with other plant promoters.All references cited herein are incorporated 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).

[0052] In the above B3), a recombinant expression vector containing the gene expression cassette can be constructed using a plant expression vector. The plant expression vector can be a Gateway system vector or a binary Agrobacterium vector, such as pGWB411, pGWB412, pGWB405, pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa or pCAMBIA1391-Xb. When using TaHsfC2aL to construct a recombinant expression vector, any enhanced, constitutive, tissue-specific or inducible promoter can be added before its transcription initiation nucleotide, such as cauliflower mosaic virus (CAMV) 35S promoter, ubiquitin gene Ubiqutin promoter (pUbi), etc., which can be used alone or in combination with other plant promoters; in addition, when using the gene of the present invention to construct a plant expression vector, enhancers can also be used, including translation enhancers or transcription enhancers. These enhancer regions can be ATG start codons or adjacent region start codons, etc., but must be the same as the reading frame of the coding sequence to ensure the correct translation of the entire sequence. The sources of the translation control signal and the start codon are extensive and can be natural or synthetic. The translation initiation region can come from the transcription initiation region or the structural gene.

[0053] The microorganism described in B4) above may be Agrobacterium, and the Agrobacterium is GV3101.

[0054] In the above application, the substance that upregulates, enhances or improves the expression of the gene encoding the protein is the above-mentioned biological material.

[0055] In the above application, the plant is any one of the following F1)-F4):

[0056] F1) dicotyledonous or monocotyledonous plants;

[0057] F2) Cruciferae and Leguminosae.

[0058] In order to solve the above problems, the present application also provides a method for increasing the weight of plant seeds.

[0059] The method for increasing plant seed weight provided in the present application comprises upregulating, enhancing or increasing the plant seed weight by upregulating, enhancing or increasing the expression of the gene encoding the above-mentioned protein or the content of the above-mentioned protein.

[0060] In order to solve the above problems, the present application also provides a method for plant breeding.

[0061] The plant breeding method provided in the present application comprises up-regulating or enhancing or increasing the expression level of the coding gene of the above-mentioned protein in the target plant or the activity or content of the protein to obtain a high seed weight plant, wherein the seed weight of the high seed weight plant is higher than that of the target plant.

[0062] In the above text, the seed weight is the dry weight of the seeds.

[0063] In the above method, the upregulation, enhancement or increase of the expression level of the gene encoding the above protein or the activity or content of the above protein in the target plant is to introduce the above biological material into the target plant.

[0064] In the above, the biological material is as follows:

[0065] D1), a nucleic acid molecule encoding the above protein.

[0066] D2), an expression cassette containing the nucleic acid molecule described in D1).

[0067] D3), ​​a recombinant vector containing the nucleic acid molecule described in D1), or a recombinant vector containing the expression cassette described in D2). The recombinant vector may be as described above, and may specifically be pGWB411.

[0068] In the above, the introduction can be achieved by microbial infection. The microorganism can be Agrobacterium. The Agrobacterium is GV3101. Specifically, it can be:

[0069] 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).

[0070] The nucleic acid molecule encoding the above protein may be:

[0071] E1), the nucleic acid sequence is the DNA shown in Sequence 1;

[0072] E2) A DNA having more than 80% identity with the DNA shown in E1) and having the ability to increase plant seed weight obtained by substituting and / or deleting and / or adding nucleotide residues to the DNA described in E1).

[0073] In the above method, the plant is any one of the following H1)-H4):

[0074] H1) dicotyledonous or monocotyledonous plants;

[0075] H2) Cruciferous and Leguminosae;

[0076] Beneficial effects:

[0077] The present invention introduces the coding gene of GmSFR2 into the model plant Arabidopsis thaliana (Columbia) to obtain a transgenic GmSFR2 Arabidopsis plant that overexpresses the GmSFR2 gene. The seed weight of the transgenic GmSFR2 Arabidopsis plant is weighed with the wild-type recipient plant (Columbia) as a control, and it is found that the seed weight of the transgenic GmSFR2 Arabidopsis plant is increased compared with the wild-type Arabidopsis. It is shown that the GmSFR2 protein and the coding gene provided by the present invention play an important role in regulating the weight of plant seeds and can be used to regulate the seed weight of plants. BRIEF DESCRIPTION OF THE DRAWINGS

[0078] Figure 1 Schematic diagram of the cloning vector and plant expression vector pGWB411–GmSFR2. A is the pCR8 / GW / TOPO vector map, and B is a partial map of pGWB411-GmSFR2.

[0079] Figure 2 This is the molecular identification of GmSFR2-overexpressing Arabidopsis strains. The horizontal axis represents different strains, and the vertical axis represents the relative expression level.

[0080] Figure 3 This is a comparison of the thousand-grain weight of seeds of GmSFR2 transgenic lines and the control. The horizontal axis represents different lines, and the vertical axis represents the thousand-grain weight of seeds. DETAILED DESCRIPTION

[0081] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0082] The experimental methods in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples are commercially available unless otherwise specified.

[0083] The following examples were processed using SPSS11.5 statistical software, and the experimental results were expressed as mean ± standard deviation. One-way ANOVA test was used, and P < 0.05 (*) indicated a significant difference, P < 0.01 (**) indicated a very significant difference, and P < 0.001 (***) indicated a very significant difference.

[0084] The soybean [Glycine max (L.) Merr] in the following examples is Nannong 1138-2 (provided by the National Soybean Improvement Center of Nanjing Agricultural University, the germplasm bank of the National Soybean Improvement Center of Nanjing Agricultural University).

[0085] The expression vector pGWB411 (Tsuyoshi Nakagawa, et al., Gatway Vectors for Plant Transformation, Plant Biotechnology, 2009, 26, 275-284) in the following examples was provided by Dr. Tsuyoshi Nakagawa and can be obtained from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences with the consent of Dr. Tsuyoshi Nakagawa. The biological material is only used to repeat the relevant experiments of the present invention and cannot be used for other purposes.

[0086] The Agrobacterium GV3101 (Lee CW et al., Agrobacterium tumefaciens promotes tumor induction by modulating pathogen defense in Arabidopsisthaliana, Plant Cell, 2009, 21(9), 2948-62) in the following examples is publicly available from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences. This biological material is only used to repeat the relevant experiments of the present invention and cannot be used for other purposes.

[0087] Example 1. cDNA cloning of the soybean seed weight-related protein GmSFR2 gene (GmSFR2) and construction of a plant expression vector

[0088] Using recombinant inbred lines of Kefeng 1 (small grain) and Nannong 1138-2 (large grain) as the parents as the mapping population, QTLs related to grain weight were located on chromosomes, including the QTL region on chromosome 17. Among the genes involved in the QTL region on chromosome 17, a gene with the locus Glyma.17G037100 (Locus name: Glyma.17G037100) was screened, encoding a glycoside hydrolase superfamily protein, as a candidate gene for regulating grain weight. After investigation, the gene responded to freezing and was related to freezing tolerance, so it was named GmSFR2. There have been no reports on the correlation between this protein and this type of enzyme and the regulation of seed weight.

[0089] Total RNA was extracted from the seedlings of Nannong 1138-2, and the RNA was reverse transcribed into cDNA using reverse transcriptase.

[0090] Primers were designed based on the information of the soybean genome sequence in PlantGDB and the GmSFR2 full-length cDNA sequence in the determined Nannong 1138-2 genome sequence. The primer sequences are as follows:

[0091] GmSFR2-up: 5'-ATGACGGTGGTCGGACTCTT

[0092] GmSFR2-dp: 5'-AGTTTCAAGAGGCTGGAGAATCA

[0093] Using Nannong 1138-2 cDNA as a template, GmSFR2-up and GmSFR2-dp as primers, and Taq DNA polymerase for PCR amplification, a PCR product of about 1.9 kb was obtained. After sequencing, the PCR product was 1914 bp (referred to as fragment 1), and its sequence was the nucleotides shown at the 1st to 1911th positions of sequence 1, sequence 1 was the CDS sequence of the GmSFR2 gene, the coding sequence of GmSFR2 was sequence 1 in the sequence table, the protein encoded by the gene was GmSFR2, and the amino acid sequence of the GmSFR2 protein was sequence 2 in the sequence table.

[0094] Gene cloning uses the Gateway system provided by Invitrogen, and the 3′-T overhang of the vector is used to directly connect the PCR product amplified by Taq enzyme. Using the principle of TA cloning, the 1914bp PCR product amplified above was connected to the cloning vector pCR8 / GW / TOPO (Invitrogen product, Figure 1 A), and the recombinant vector pTOPO-GmSFR2 was obtained. Both pTOPO-GmSFR2 and the target expression vector pGWB411 carry the recombination sites attR1 and attR2. The pTOPO-GmSFR2 containing GmSFR2 and the expression vector pGWB411 (GenBank: AB294435.1, 24-AUG-2007) were subjected to LR recombination reaction under the action of recombinase, and the target gene GmSFR2 was successfully constructed into the expression vector pGWB411. The obtained recombinant vector was named pGWB411-GmSFR2 ( Figure 1 (middle B).

[0095] pGWB411-GmSFR2 is a recombinant expression vector obtained by replacing the fragment between the attR1 site and the attR2 site of the pGWB411 vector with attB1-GmSFR2-attB2, while keeping the other nucleotides of pGWB411 unchanged.

[0096] Example 2: Obtaining Arabidopsis thaliana overexpressing GmSFR2

[0097] 1. Obtaining recombinant Agrobacterium

[0098] The recombinant vector pGWB411-GmSFR2 containing GmSFR2 obtained in Example 1 was introduced into Agrobacterium GV3101 by electroporation to obtain recombinant Agrobacterium containing pGWB411-GmSFR2, which was named recombinant Agrobacterium GV3101 / GmSFR2.

[0099] 2. Obtaining and Identifying GmSFR2 Transgenic Arabidopsis

[0100] The recombinant Agrobacterium GV3101 / GmSFR2 was cultured to the logarithmic phase and then transformed into Columbia ecotype Arabidopsis thaliana (Col-0) (seeds from Arabidopsis Biological Resource Center (ABRC)) by vacuum method. After cultivation, the seeds were harvested (T 1 The seeds were sown on MS screening medium containing kanamycin (50 mg / L) and the T 1 When the first generation plants grow to 4-6 leaves, they are moved to vermiculite for growth and harvested. 1 Generation of individual plants, each individual plant seed (T 2 The same MS screening medium was used to continue screening to observe the T 2 This process was repeated for several generations until a genetically stable homozygous transgenic line was obtained. Nine GmSFR2 transgenic Arabidopsis pure lines (T 4 The expression level of GmSFR2 gene was detected by randomly selecting three strains named OE1, OE15 and OE26 from the nine strains.

[0101] Total RNA of seedlings of the above three strains and Columbia ecotype Arabidopsis (Col-0, as wild-type Arabidopsis control, referred to as control) was extracted and reverse transcribed. The cDNA obtained by reverse transcription was used as template, and the primers were: GmSFR2-up: 5'-ATGACGGTGGTCGGACTCTT and GmSFR2-dp: 5'-AGTTTCAAGAGGCTGGAGAATCA, and Real Time-PCR identification was performed. Arabidopsis AtActin2 gene was used as internal standard, and the primers used were Primer-TF: 5'-ATGCCCAGAAGTCTTGTTCC, and Primer-TR: 5'-TGCTCATACGGTCAGCGATA-3'. The expression level of the internal standard AtActin2 gene was taken as 1, and the relative expression level of GmSFR2 encoding gene GmSFR2 was determined. The experiment was repeated three times, and the results were averaged. The relative expression levels of GmSFR2 in OE1, OE15, and OE26 were 0.002, 0.0015, and 0.11, respectively. The expression level of GmSFR2 was not detected in the wild-type Arabidopsis control ( Figure 2 ).

[0102] The above results further proved that GmSFR2 was transferred into Arabidopsis and expressed. The three strains OE1, OE15 and OE26 were GmSFR2 overexpression strains.

[0103] 3. Phenotypic analysis of GmSFR2-transgenic Arabidopsis

[0104] First, the phenotypes of the control and three GmSFR2 overexpression lines, OE1, OE15 and OE26, under normal conditions were tested. Under normal conditions, the phenotypes of the control and GmSFR2 overexpression lines, such as rosette and plant height, were not significantly different from those of the control.

[0105] The thousand-grain weight of seeds of GmSFR2 overexpressing lines OE1, OE15 and OE26 was measured using wild-type Arabidopsis Col as a control. The specific operation was as follows: Arabidopsis seeds at maturity were harvested, dried at room temperature for 7 days, weighed, and then dried for another 5 days and weighed until the weight remained constant.

[0106] Seeds of 20 plants were collected from each strain of wild-type Arabidopsis thaliana Col, OE1, OE15 and OE26, and 200 seeds were weighed for each strain, and the thousand-grain weight data of the seed of the strain were obtained by weighing with a precision balance. The biological experiment was repeated three times, and the results were taken as the mean ± standard deviation.

[0107] The results are as follows Figure 3 As shown, the thousand-grain weights of wild-type Arabidopsis Col, GmSFR2 overexpression lines OE1, OE15 and OE26 were 16.7±2.3, 23.4±6.9, 23.6±3.2 and 20.1±2.1 mg, respectively. The results showed that the thousand-grain weights of the three GmSFR2 overexpression transgenic lines were significantly or extremely significantly higher than those of the wild-type control ( Figure 3 ). Figure 3 In the table, * indicates significant difference compared with wild-type Arabidopsis; ** indicates extremely significant difference compared with wild-type Arabidopsis.

[0108] The above experiment shows that overexpression of GmSFR2 increases the thousand-grain weight of transgenic plant seeds, and while increasing the seed weight (grain weight) of transgenic plants, it does not affect the normal growth of the plants. Therefore, this gene can be used as a target gene to increase plant seed yield. GmSFR2 is a protein related to seed weight.

[0109] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. Application of relevant biomaterials in any of the following; The biological material is any of the following: D1), nucleic acid molecules encoding proteins; 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); The application is any of the following: A1) Increase plant seed weight; A2) preparing a product for increasing the weight of plant seeds; A3) Cultivating high seed weight plants; A4) preparing and cultivating high-grain-weight seed plant products; The protein is a protein whose amino acid sequence is shown in Sequence 2, and the plant is a plant of the Cruciferae family and the Leguminosae family.

2. The use according to claim 1, characterized in that The coding gene of the protein is the DNA whose nucleic acid sequence is shown in Sequence 1.

3. A method for increasing the weight of plant seeds, characterized in that: The method comprises introducing the biological material of claim 1 or 2 into the target plant to increase the seed weight of the target plant, and the plant is a plant of the cruciferous family and the leguminous family.

4. A method for plant breeding, characterized in that: The method comprises introducing the biological material of claim 1 or 2 into the target plant to obtain a high seed weight plant, wherein the seed weight of the high seed weight plant is higher than that of the target plant, and the plant is a cruciferous plant or a leguminous plant.

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