Soybean photosynthesis-related gene GmGRF5-2 and its encoded protein and its application

Abstract

The present invention relates to the field of genetic engineering, and in particular to a soybean photosynthesis-related gene GmGRF5‑2, its encoded protein and application. The present invention provides a soybean GmGRF5‑2 protein, which is: 1) a protein consisting of the amino acid sequence shown in SEQ ID NO.1, or 2) a protein derived from 1) that has equivalent activity by replacing, deleting or adding one or more amino acids in the amino acid sequence shown in SEQ ID NO.1. The present invention also provides a gene GmGRF5‑2 encoding the above-mentioned protein, the nucleotide sequence of which is shown in SEQ ID NO.2. Overexpression of the GmGRF5‑2 gene can promote photosynthesis, increase 100-grain weight, improve soybean quality, and increase the protein content of soybean seeds, and has potential application value in agricultural production.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering, and in particular to a soybean photosynthesis-related gene GmGRF5-2, its encoded protein and application. Background Art

[0002] Paths to increasing crop yields typically include breeding high-yield varieties, applying organic fertilizers and yield-increasing agents, and improving cultivation and farming practices. These methods include traditional hybridization and modern molecular breeding (including transgenic breeding). Transgenic breeding, based on a clear understanding of the target gene's function and mechanism of action, results in a shorter cycle and higher yield-increasing efficiency. Numerous reports have also been published on soybeans using genetic modification to increase yield and resistance. For example, soybeans overexpressing the Ncl gene (Na+, K+, and Cl- transporter) have higher salt tolerance and yields 3.6-5.5 times higher than wild-type soybeans in high-salt areas (Do et al., 2016). Transgenic soybeans expressing LOS5 / ABA3 (abscisic acid aldehyde dehydrogenase activity-related gene) have reduced stomatal opening size and transpiration rate under drought conditions, significantly enhancing drought resistance and increasing yield by approximately 21% compared to wild-type soybeans under drought conditions (Li et al., 2013). Experiments in the United States and Argentina have shown that overexpressing sunflower Hahb-4 (ethylene signal transduction pathway gene) in soybeans can delay soybean senescence and increase yield by 7-15% under drought and other adverse conditions (Waltz et al., 2013). Monsanto reported that overexpressing the Arabidopsis thaliana BBX32 flowering gene in soybeans increased yield by 5-7% (Preuss et al., 2012). Overexpression of cyanobacterial FBP / SBPase (Bifunctional Soybeans expressing the APETALA2-like gene (GmTOE4a) maintain stable yields under stresses such as high CO2 concentrations and high temperatures (i.e., potential future environmental conditions) (Kohler et al., 2016). Downregulating the expression of the soybean BS1 (Big Seed 1) homolog significantly increases soybean seed size, with dry weight increasing by over 45% (Ge et al., 2016). Transgenic soybeans overexpressing the APETALA2-like gene GmTOE4a exhibit agronomic traits with high yield potential, such as shorter plant height and internodes, and stronger stems (Zhao et al., 2016).

[0003] As the strongest plant-based protein provider, soybeans provide a major source of protein for humans and livestock. Using genetic engineering to introduce protein-enhancing genes into commercial varieties will significantly increase the protein supply for livestock and humans worldwide. Even a one-percentage-point increase in soybean protein yields millions of tons of additional protein.

[0004] Soybean seed proteins can be divided into three categories based on their biological functions: storage proteins, structural proteins, and defense-related proteins. Storage proteins predominate and are one of the most important plant proteins for human consumption. Storage proteins are primarily composed of glycinins and conglycinins. To date, seven homologous genes encoding glycinins have been cloned and sequenced: Gy1, Gy2, Gy3, Gy4, Gy5, Gy6, and Gy7 (Chen Jinling, 2020). Reports on other genes regulating the function of soybean seed protein content are less common.

[0005] Growth-regulating factors (GRFs) play an important role in regulating plant growth and development. Currently, research on the functions of GRFs has mainly focused on plants such as Arabidopsis, rice, and corn, while few studies on the functions of this family of genes in soybean have been reported. Summary of the Invention

[0006] The purpose of the present invention is to provide a soybean photosynthesis-related gene GmGRF5-2, the protein encoded by the gene, and its application in regulating plant photosynthesis, 100-grain weight and seed protein accumulation.

[0007] In a first aspect, the present invention provides a soybean GmGRF5-2 protein, wherein the amino acid sequence of the soybean GmGRF5-2 protein comprises:

[0008] 1) the amino acid sequence shown in SEQ ID NO.1, or

[0009] 2) An amino acid sequence derived from 1) having equivalent activity, wherein one or more amino acids are substituted, deleted or added to the amino acid sequence shown in SEQ ID NO. 1.

[0010] It should be understood that those skilled in the art can substitute, delete, and / or add one or more amino acids based on the amino acid sequence disclosed in the present invention without affecting its activity to obtain a mutant sequence of the protein. Therefore, the soybean GmGRF5-2 protein of the present invention also includes proteins derived from the soybean GmGRF5-2 protein that have the same activity as the soybean GmGRF5-2 protein by substituting, replacing, and / or adding one or more amino acids to the amino acid sequence shown in SEQ ID No. 1.

[0011] In a second aspect, the present invention provides a gene encoding the soybean GmGRF5-2 protein. More specifically, the soybean GmGRF5-2 protein gene provided by the present invention has a nucleotide sequence as shown in SEQ ID NO. 2 or a nucleotide sequence having 95% or greater homology to the nucleotide sequence as shown in SEQ ID NO. 2.

[0012] The GmGRF5-2 (full name: GROWTH-REGULATING FACTOR) gene has the nucleotide sequence shown in SEQ ID No. 2 or a nucleotide sequence having 95% or greater homology to the nucleotide sequence shown in SEQ ID No. 2. The soybean GmGRF5-2 gene of the present invention was cloned from soybean Tianlong No. 1 by RT-PCR.

[0013] The gene of the present invention includes a nucleic acid sequence encoding the protein. In addition, it should be understood that, taking into account the degeneracy of codons and the preference of codons of different species, those skilled in the art can use codons suitable for expression of specific species as needed.

[0014] In a third aspect, the present invention provides a biological material carrying the GmGRF5-2 gene, wherein the biological material is an expression cassette, an expression vector, a host cell or a host bacteria.

[0015] The plant expression vector of the present invention is pSoy1.

[0016] The present invention constructs the GmGRF5-2 gene into the expression vector pSoy1 and propagates it in Escherichia coli DH5α. Using Agrobacterium-mediated transformation, the GmGRF5-2 gene carried by pSoy1 is transferred into soybean Tianlong No. 1, generating transgenic soybeans overexpressing GmGRF5-2. Results demonstrate that GmGRF5-2 enhances plant photosynthesis, increases 100-seed weight, and increases seed protein content.

[0017] The present invention also provides cloning vectors or various expression vectors containing the GmGRF5-2 nucleotide sequence or a fragment thereof, host cells containing the vector, transformed plant cells containing the nucleotide sequence or a specific fragment thereof, and transgenic plants.

[0018] Therefore, according to the understanding of those skilled in the art, the present invention seeks protection for:

[0019] Application of soybean GmGRF5-2 protein or its encoding gene or biological materials containing the encoding gene in regulating plant photosynthesis, increasing 100-grain weight or improving soybean quality.

[0020] Application of soybean GmGRF5-2 protein or its encoding gene or biological materials containing the encoding gene in plant germplasm resource improvement or hybrid breeding.

[0021] Application of soybean GmGRF5-2 protein or its encoding gene or biological material containing the encoding gene in preparing transgenic plants.

[0022] In the above application provided by the present invention, the transgenic plant is a transgenic plant with increased photosynthesis rate and / or increased 100-grain weight and / or increased seed protein content relative to wild-type plants.

[0023] It's important to note that soybean photosynthesis occurs during the vegetative growth phase. The division between vegetative and reproductive growth is usually marked by flower bud differentiation, with the period before this being considered vegetative and the period after this being considered reproductive. However, there's no strict boundary between these two phases. For a considerable period, these two phases occurred simultaneously, with significant competition for nutrients.

[0024] The physiological processes of oil and protein synthesis in plant seeds are highly complex. Proteins, in particular, contain a variety of amino acids, most of which have their own distinct synthesis pathways involving numerous genes and enzymes. Oil composition and accumulation are influenced by the activities of multiple enzymes in the fatty acid synthesis pathway. The expression of these genes is also regulated at the pre-, transcriptional, and post-transcriptional levels, with numerous genes involved in this process.

[0025] Therefore, it is unpredictable what role the photosynthesis-related protein GmGRF5-2 will play in the synthesis or accumulation of seed proteins in the late reproductive growth stage of plants.

[0026] The present invention also provides a method for preparing transgenic plants with increased photosynthesis rate and / or increased 100-grain weight and / or increased protein content in seeds, which comprises introducing or overexpressing the soybean GmGRF5-2 gene of the present invention into the plant genome.

[0027] The beneficial effects of the present invention are:

[0028] (1) Provided are the soybean GmGRF5-2 gene and its encoded protein GmGRF5-2;

[0029] (2) Overexpression of the GmGRF5-2 gene in plants can promote photosynthesis, increase 100-grain weight, and improve seed protein content. Therefore, the GmGRF5-2 gene and its encoded protein can promote plant photosynthesis, increase 100-grain weight, and improve seed protein content. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The present invention compares the amino acid sequence of the protein encoded by the soybean photosynthesis-related gene GmGRF5-2 with the amino acid sequence of the protein encoded by the Arabidopsis GRF5 gene.

[0031] Figure 2 Schematic diagram of the structure of the cloning intermediate vector pGWCm in Example 2 of the present invention.

[0032] Figure 3Schematic diagram of the structure of the plant expression vector pSoy1 of Example 3 of the present invention.

[0033] Figure 4 Schematic diagram of the structure of the CRISPR-Cas9 expression vector of Example 4 of the present invention.

[0034] Figure 5 In Example 5 of the present invention, overexpression of the soybean photosynthesis-related gene GmGRF5-2 in soybean transformation resulted in increased photosynthetic rate (left) and chlorophyll content (right). Conversely, the CRISPR-Cas9 mutant exhibited decreased photosynthetic rate (left) and chlorophyll content (right). TL in the figure represents Tianlong No. 1, and WS82 represents Williams 82.

[0035] Figure 6 In Example 6 of the present invention, soybean transformation by overexpressing the soybean photosynthesis-related gene GmGRF5-2 resulted in a significant increase in 100-grain weight, while the 100-grain weight of its mutant was significantly reduced; in the figure, TL is Tianlong No. 1 and WS82 is Williams 82.

[0036] Figure 7 The soybeans transformed by overexpressing the soybean photosynthesis-related gene GmGRF5-2 in Example 7 of the present invention resulted in a significant increase in seed protein content. Conversely, the seed protein content of the mutant was significantly reduced. In the figure, TL represents Tianlong No. 1 and WS82 represents Williams 82.

[0037] Figure 8 Transformation of soybeans by overexpressing the soybean photosynthesis-related gene GmGRF5-2, as described in Example 8 of the present invention, resulted in a significant reduction in seed oil content. Seed oil content in this mutant was also significantly reduced. In the figure, TL represents Tianlong No. 1 and WS82 represents Williams 82. DETAILED DESCRIPTION

[0038] The following examples are intended to illustrate the present invention but are not intended to limit the scope of the present invention. Without departing from the spirit and substance of the present invention, modifications or substitutions made to the methods, steps or conditions of the present invention are intended to fall within the scope of protection of the present invention.

[0039] Unless otherwise specified, the experimental materials, reagents, instruments, etc. used in the examples of the present invention are all commercially available; unless otherwise specified, all technical means in the examples of the present invention are conventional means well known to those skilled in the art.

[0040] Example 1 Cloning of soybean photosynthesis-related gene GmGRF5-2

[0041] This example provides a method for cloning the soybean photosynthesis-related gene GmGRF5-2, and the steps are as follows:

[0042] The GmGRF5-2 gene was cloned and sequenced from Glycine max L. Tianlong 1 using the forward primer 5'-ATGATGAGTGCAAGTGCAGGTGC-3' (SEQ ID NO.3) and the reverse primer 5'-TCATTCATCGGTTTGGATTCTGG-3' (SEQ ID NO.4), respectively. The gene sequence is shown in SEQ ID NO.2; the amino acid sequence of the protein encoded by it is shown in SEQ ID NO.1.

[0043] The PCR reaction program was as follows: pre-denaturation at 95°C for 5 min, 25 cycles of 94°C for 30 s, 55°C for 35 s, and 72°C for 1 min 30 s, and extension at 72°C for 10 min.

[0044] The amino acid sequence of the soybean photosynthesis-related gene GmGRF5-2 is 45.3% similar to that of the Arabidopsis GRF5 protein. Figure 1 .

[0045] Example 2 Cloning vector of soybean photosynthesis-related gene GmGRF5-2

[0046] This example provides a method for obtaining a cloning vector for the soybean photosynthesis-related gene GmGRF5-2, comprising the following steps:

[0047] The PCR product obtained from Example 1 was directly cloned into Figure 2 The pGWCm vector shown in Figure 1 was first hydrolyzed with Ahd I endonuclease, and the digestion product was recovered using a gel extraction kit to obtain the T vector. The PCR product and the T vector were then ligated at 16°C. The ligated product was transformed into E. coli DH5α, amplified, and positive clones were screened and sequenced.

[0048] Example 3 Plant expression vector of soybean photosynthesis-related gene GmGRF5-2

[0049] This example provides a method for preparing a plant expression vector of the soybean photosynthesis-related gene GmGRF5-2, comprising the following steps:

[0050] The cloning vector of soybean photosynthesis-related gene GmGRF5-2 obtained in Example 2 was cloned into Figure 3The plant expression vector pSoy1 shown below was mixed in equal proportions and then transformed using an LR reaction (50 ng of each plasmid, 1 μl of LR enzyme, and HO added to a final volume of 5 μl. Mix thoroughly and incubate at 25°C for at least 6 hours). This allowed the construction of GmGRF5-2, a soybean photosynthesis-related gene, in plants to overexpress it and study its function. Plant transformation was performed using Agrobacterium-mediated transformation. The selection marker in plants was Bar.

[0051] Example 4 CRISPR-Cas9 vector of soybean photosynthesis-related gene GmGRF5-2

[0052] This example provides a method for constructing a CRISPR-Cas9 vector for the soybean photosynthesis-related gene GmGRF5-2, and the steps are as follows:

[0053] First, the entry vectors expressing Cas9 protein and gRNA were constructed separately. The Cas9 gene driven by the 35S promoter was digested and ligated into the vector Fu76 via the Stu I and Mfe I cloning sites to obtain the entry vector Fu76-Cas9 (containing the attL3 / 4 recombination sites). The GmU6 promoter, two tandem Bsa I sites (SEQ ID NO.5: AgagaccAAggtctcA) and the gRNA scaffold fragment were concatenated, and the fragment was enzymatically ligated between Hind III and Sph I of the Fu79 vector; the second group of fragments (GmU6 promoter, two tandem BfuA I sites SEQ ID NO.6: ACCAgcaggtAAacctgcCACA) and the gRNA scaffold fragment were enzymatically ligated between Kpn I and Sph I of the Fu79 vector; the third group of fragments (GmU6 promoter, two tandem BspQ I sites (SEQ ID NO.7: ATTAgaagagcAAgctcttcA) and the gRNA scaffold fragment were enzymatically ligated to the Fu79 vector) to obtain the entry vector Fu79-gRNA (containing attL1 / 2 recombination sites) expressing three gRNAs.

[0054] In the present invention, GmGRF5-2 was used as the target gene, sgRNA was designed using the CRISPR-P tool (http: / / crispr.hzau.edu.cn / cgi-bin / CRISPR2 / CRISPR), and three target sequences with higher scores were selected as the editing sites of CRISPR-Cas9. Then, the three target sequence fragments were artificially synthesized and annealed by primers and connected to the Fu79-s gRNA vector using BsaI, BfuA I, and BsQ I, respectively. The two entry vectors were cloned into the pSoy2 expression vector by LR reaction to obtain the pSoy2-CAS9-gRNA1-gRNA2-gRNA3 expression vector for GmGRF5-2 gene editing ( Figure 4 After the vector was transformed into Agrobacterium EHA105, gene-edited plants were obtained through the soybean cotyledonary node transformation method.

[0055] Example 5: Soybean photosynthesis-related gene GmGRF5-2 promotes soybean photosynthesis

[0056] In this example, the soybean transformation method of Wang Kan et al. (Paz, M., Wang, K. Soybean transformation and regeneration using half-seed explants. US Patent #7,473,822 (Issued January 6, 2009)) was used to obtain transgenic soybeans overexpressing 35S:GmGRF5-2:HA, and a mutant of GmGRF5-2 was obtained using the CRIPR-Cas9 method. Figure 5 Transformation of soybeans with the photosynthesis-related gene GmGRF5-2 results in increased photosynthetic rate and chlorophyll content. The left figure shows the photosynthetic rate of wild-type Tianlong 1 (TL), Williams 82 (WS82), overexpression, and mutant soybeans. The right figure shows the chlorophyll content of wild-type Tianlong 1 (TL), Williams 82 (WS82), overexpression, and mutant soybeans. The results of soybean photosynthetic rate and chlorophyll content are shown in Table 1.

[0057] Table 1 Photosynthetic rate and chlorophyll content of soybean

[0058] TL GmGRF5-2:HA#2 WS82 Gmgrf-2#1 photosynthetic rate 16.07±0.49 18.51±1.04* 24.47±1.60 21.29±1.60* Chlorophyll content 27.65±5.40 41.94±3.65* 44.62±1.42 41.75±1.34*

[0059] Example 6: Soybean photosynthesis-related gene GmGRF5-2 promotes increase in 100-grain weight

[0060] The 100-grain yield of wild type, GmGRF5-2 overexpression and mutant was determined as shown in Table 2. Figure 6 ,from Figure 6 The results showed that overexpression of GmGRF5-2 can increase the 100-grain weight of soybean seeds, while the 100-grain weight of the mutant was significantly reduced. The results of single-plant yield determination showed that overexpression of GmGRF5-2 can increase yield, while the yield of the mutant was significantly reduced.

[0061] Table 2 Results of GmGRF5-2 in improving soybean yield

[0062] TL GmGRF5-2:HA#2 WS82 Gmgrf-2#1 100-grain weight (g) 14.24±0.12 15.91±0.11*** 18.59±0.99 16.32±0.36** Yield per plant (g) 4.48±0.84 4.78±1.99 5.64±1.01 4.46±1.00*

[0063] Example 7: Soybean photosynthesis-related gene GmGRF5-2 increases seed protein content

[0064] This example explores the application of soybean photosynthesis-related gene GmGRF5-2 in increasing seed protein content. By measuring the protein content in wild-type, GmGRF5-2 overexpression and mutant seeds, this example found that overexpression of GmGRF5-2 can increase the protein content in soybean seeds, while the protein content in soybean seeds of mutants decreased, indicating that GmGRF5-2 has the function of promoting protein accumulation in seeds. Figure 7 and Table 3.

[0065] Table 3 Results of GmGRF5-2 increasing soybean seed protein content

[0066]

[0067] Example 8 Correlation between soybean photosynthesis-related gene GmGRF5-2 and seed oil content

[0068] This example explores the changes in oil content in the above-mentioned overexpressed transgenic materials and mutant seeds. Figure 8 As shown in Table 4 , overexpression of GmGRF5-2 reduced the oil content in seeds, and mutation also resulted in a decrease in oil content in seeds.

[0069] Table 4 Results of GmGRF5-2 reducing soybean seed oil content

[0070] TL GmGRF5-2:HA#2 WS82 Gmgrf-2#1 Seed oil content (%) 18.01±0.28 17.58±0.29* 21.47±0.39 20.892±0.56*

[0071] Although the present invention has been described in detail above using general descriptions and specific embodiments, it will be apparent to those skilled in the art that modifications and improvements may be made based on the present invention. Therefore, such modifications and improvements, which do not depart from the spirit of the present invention, are intended to be within the scope of protection claimed herein. Sequence Listing <110> Institute of Crop Sciences, Chinese Academy of Agricultural Sciences <120> Soybean photosynthesis-related gene GmGRF5-2 and its encoded protein and its application <130> KHP221113960.5 <160> 7 <170> SIPOSequenceListing 1.0 <210> 1 <211> 359 <212> PRT <213> Artificial Sequence <400> 1 Met Met Ser Ala Ser Ala Gly Ala Arg Asn Arg Ser Pro Phe Thr Gln 1 5 10 15 Thr Gln Trp Gln Glu Leu Glu Gln Gln Ala Leu Val Phe Lys Tyr Met 20 25 30 Val Thr Gly Thr Pro Ile Pro Pro Asp Leu Ile Tyr Ser Ile Lys Arg 35 40 45 Ser Leu Asp Thr Ser Ile Ser Ser Arg Leu Phe Pro His His Pro Ile 50 55 60 Gly Trp Gly Cys Phe Glu Met Gly Phe Gly Arg Lys Val Asp Pro Glu 65 70 75 80 Pro Gly Arg Cys Arg Arg Thr Asp Gly Lys Lys Trp Arg Cys Ser Lys 85 90 95 Glu Ala Tyr Pro Asp Ser Lys Tyr Cys Glu Arg His Met His Arg Gly 100 105 110 Arg Asn Arg Ser Arg Lys Pro Val Glu Val Ser Ser Ala Thr Ser Thr 115 120 125 Ala Thr Asn Thr Ser Gln Thr Ile Pro Ser Ser Tyr Thr Arg Asn Leu 130 135 140 Ser Leu Thr Asn Asn Ser Asn Pro Asn Ile Thr Pro Pro Pro Pro Pro 145 150 155 160 Ser Ser Phe Pro Phe Ser His Leu Pro Ser Ser Met Pro Ile Asp Gln 165 170 175 Ser Gln Pro Phe Ser Gln Ser Tyr Gln Asn Ser Ser Leu Asn Pro Phe 180 185 190 Phe Tyr Ser Gln Ser Thr Ser Ser Arg Pro Pro Asp Ala Asp Phe Pro 195 200 205 Pro Gln Asp Ala Thr Thr His His Leu Phe Met Asp Ser Ala Gly Ser 210 215 220 Tyr Ser His Asp Glu Lys Asn Tyr Arg His Val His Gly Ile Arg Glu 225 230 235 240 Asp Val Asp Glu Arg Ala Phe Phe Pro Glu Ala Ser Gly Ser Ala Arg 245 250 255 Ser Tyr Thr Asp Ser Tyr Gln Gln Leu Ser Met Ser Ser Tyr Lys Ser 260 265 270 Tyr Ser Asn Ser Asn Phe Gln Asn Ile Asn Asn Asp Ala Thr Thr Asn 275 280 285 Pro Arg Gln Gln Glu Gln Gln Leu Gln Gln Gln Gln His Cys Phe Val 290 295 300 Leu Gly Thr Asp Phe Lys Ser Thr Arg Pro Ser Lys Glu Lys Glu Ala 305 310 315 320 Glu Thr Thr Thr Gly Gln Arg Pro Leu His Arg Phe Phe Gly Glu Trp 325 330 335 Pro Pro Lys Asn Thr Thr Thr Asp Ser Trp Leu Asp Leu Ala Ser Asn 340 345 350 Ser Arg Ile Gln Thr Asp Glu 355 <210> 2 <211> 1080 <212> DNA <213> Artificial Sequence <400> 2 atgatgagtg caagtgcagg tgcaagaaat aggtctccgt tcacacaaac tcagtggcaa 60 gagcttgagc aacaagctct tgtttttaag tacatggtta caggaacacc tatcccacca 120 gatctcatct actctattaa aagaagtcta gacacttcaa tttcttcaag gctcttccca 180 catcatccaa ttgggtgggg atgttttgaa atgggatttg gcagaaaagt agacccagag 240 ccagggaggt gcagaagaac agatggcaag aaatggagat gttcaaagga ggcatatcca 300 gactcaaagt actgtgaaag acacatgcac agaggcagaa accgttcaag aaagcctgtg 360 gaagtttctt cagcaacaag caccgccaca aacacctccc aaacaatccc atcatcttat 420 accagaaacc tttccttgac caataacagt aaccccaaca taacaccacc accaccaccc 480 tcttctttcc ctttctctca tttgccctct tctatgccta ttgatcagtc ccaacccttt 540 tcccaatcct accaaaactc ttctctcaat cccttcttct actcccaatc aacctcctct 600 agacccccag atgctgattt tccaccccaa gatgccacca cccaccacct attcatggac 660 tctgctggct cttattctca tgatgaaaag aattataggc atgttcatgg aataagggaa 720 gatgtggatg agagagcttt cttcccagaa gcatcaggat cagctaggag ctatacagac 780 tcgtaccaac aactatcaat gagctcctac aagtcctatt caaactccaa ctttcagaac 840 attaataatg atgccaccac caacccaaga cagcaagagc agcaactaca acaacaacaa 900 cactgttttg ttttagggac agacttcaaa tcaacaaggc caagcaaaga gaaagaagct 960 gagacaacaa caggtcagag accccttcac cgtttctttg gggagtggcc accaaagaac 1020 acaacaacag attcctggct agatcttgct tccaactcca gaatccaaac cgatgaatga 1080 <210> 3 <211> twenty three <212> DNA <213> Artificial Sequence <400> 3 atgatgagtg caagtgcagg tgc 23 <210> 4 <211> twenty three <212> DNA <213> Artificial Sequence <400> 4 tcattcatcg gtttggattc tgg 23 <210> 5 <211> 16 <212> DNA <213> Artificial Sequence <400> 5 agagaccaag gtctca 16 <210> 6 <211> twenty two <212> DNA <213> Artificial Sequence <400> 6 accagcaggt aaacctgcca ca 22 <210> 7 <211> twenty one <212> DNA <213> Artificial Sequence <400> 7 attagaagag caagctcttc a 21

Claims

1. Application of soybean GmGRF5-2 protein or its encoding gene in increasing soybean 100-grain weight and improving soybean seed protein content; The amino acid sequence of the soybean GmGRF5-2 protein is shown in SEQ ID NO.

1.

2. Application of soybean GmGRF5-2 protein or its encoding gene in soybean germplasm improvement or hybrid breeding; The amino acid sequence of the soybean GmGRF5-2 protein is shown in SEQ ID NO.1; The purpose of soybean germplasm resource improvement or hybrid breeding is to increase soybean 100-grain weight and improve seed protein content.

3. Use of soybean GmGRF5-2 protein or its encoding gene in the preparation of transgenic soybeans; The amino acid sequence of the soybean GmGRF5-2 protein is shown in SEQ ID NO.1; The transgenic soybean has higher 100-grain weight and seed protein content.

4. A method for preparing transgenic soybeans with increased 100-grain weight and improved seed protein content, characterized in that: A gene encoding a protein with an amino acid sequence as shown in SEQ ID NO. 1 or a gene with a nucleotide sequence as shown in SEQ ID NO. 2 is introduced into or overexpressed in the plant genome.

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