Cloning and application of genes regulating soybean seed size

Abstract

The present invention provides an isolated gene for regulating soybean grain weight, wherein the coding region sequence of the gene is shown in SEQ ID NO: 1 or SEQ ID NO: 2 or a homologous sequence thereof. The present invention also provides a method for cultivating soybeans with increased yield, which comprises transforming the GmSW17 gene or a vector or host cell containing the GmSW17 gene into soybean plant cells or tissues and cultivating them to obtain soybean plants with increased yield. At the same time, the present invention discloses the use of the GmSW17 gene or a vector or host cell containing the GmSW17 gene in cultivating soybeans with increased yield. The present invention will have great theoretical and application value for soybean breeding and related application research.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to the cloning and application of genes for regulating soybean seed size, and more specifically to the cloning and application of genes for regulating soybean 100-seed weight. Background Art

[0002] Soybean (Glycine max[L].Merr.) is one of the world's important oil crops and cash crops, and is also the main source of high-quality protein and feed protein for humans. Soybean is native to my country, but there is a serious contradiction between supply and demand of soybean in my country. Currently, more than 85% of consumption depends on imports. The main reason is that my country's soybean yield is low, so it is urgent to increase my country's soybean yield.

[0003] Soybean yield depends largely on grain weight, i.e., grain size (including grain length, width, and thickness). However, so far, the cloning of genes regulating soybean seed size is still relatively limited, and its genetic basis and specific regulatory mechanism in soybean germplasm resource variation are not clear. Therefore, in-depth exploration of the molecular genetic mechanism regulating soybean seed size is of great significance to improving soybean yield.

[0004] DUBs (deubiquitinating enzymes) are deubiquitinating proteases that can cleave ubiquitin or ubiquitin-like proteins from conjugates of pro- or target proteins, and they play multiple roles in the ubiquitin pathway (Reyes-Turcu et al., 2009). Based on the similarity of sequence and structure, deubiquitinases can be divided into seven families: ubiquitin-specific proteases (USPs), ubiquitin C-terminal hydrolases (UCHs), ovarian tumor proteases (OTUs), Machado-Joseph disease proteases (MJDs), MINDY protease family (MIU-containing novel DUB family), JAMM family (Jad1 / Pad / MPN domain-containing metallo enzymes) and the newly discovered ZUFSP / Mug105 family (zinc finger with UFM1-specific peptidase domain protein / C6orf113 / ZUP1) (Haahr et al., 2018; Wei Dong, 2019). Except for the JAMM family, which is a zinc-dependent metalloprotease, all DUBs belong to cysteine ​​proteases (Wu et al., 2019). DUBs recognize substrates anchored with monoubiquitin and polyubiquitin chains and selectively cleave them. The functions of deubiquitinases in cells can be roughly divided into the following categories: (1) processing ubiquitin precursors to produce free ubiquitin molecules; (2) removing ubiquitin chains on proteins to prevent them from being degraded by the proteasome, thereby stabilizing the proteins; (3) removing non-degradable ubiquitination signals attached to proteins; (4) ensuring the homeostasis of ubiquitin molecules in cells by preventing ubiquitin molecules from being degraded along with substrate proteins; (5) participating in the disintegration of free ubiquitin chains in cells; (6) editing the type of ubiquitin chains by cleaving ubiquitin chains (Komander et al., 2009; Wei Dong, 2019).

[0005] In plants, UBPs are the largest subfamily of DUBs. For example, in Arabidopsis, there are 27 UBP members, accounting for more than half of all DUBs. In order to cope with a variety of abiotic or biotic stresses, the growth and development of plants are strictly controlled by multiple factors throughout the growth cycle. Recent data indicate that UBPs may play a role in the entire life cycle of plants (Wu et al., 2019). UBP1 and UBP2 in Arabidopsis are homologous genes to each other and are plant-specific. In vitro, both can hydrolyze ubiquitin linked to K48. Compared with the wild type, the T-DNA insertion mutants ubp1 and ubp2 have no obvious phenotype, but in the absence of arginine and the presence of its analog canavanine, they show dwarfing, short roots, and yellow leaves, indicating that UBP1 and UBP2 play a role in the process of resistance to canavanine (Yan et al., 2000). In Arabidopsis, DA3 encodes Ubiquitin-specific protease14 (UBP14), which interacts with the inhibitor of the APC / C complex UVI4 to affect the stability of CYCA2;3 and CDKB1;1, thereby negatively regulating nuclear duplication and causing changes in organ and seed size. UBIQUITIN-SPECIFIC PROTEASE (UBP15) is a DA1 suppressor SOD2 (SUPPRESSOR OF DA1), which encodes a ubiquitin-specific protease. UBP15 regulates seed size by promoting cell proliferation in ovules and seed coats of developing seeds (Du et al., 2014). In rice, qNPT1 encodes a deubiquitinase OsOTUB1 that is homologous to human OTUB1. Unlike human OTUB1, OsOTUB1 can cleave both K48- and K63-linked polyubiquitin. Downregulation of OsOTUB1 enhances meristem activity, resulting in a decrease in tiller number, an increase in grain number and grain weight, and ultimately an increase in yield (Wang et al., 2017). No related UBP family members have been reported to be involved in grain development in soybean.

[0006] Therefore, it is of great significance to explore and study the regulatory mechanism of UBP family members on soybean grain development, which plays a very important role in improving the yield and quality of soybean. Summary of the invention

[0007] In order to further improve the yield and quality of soybean, the present invention analyzed the genetic regulatory sites of complex yield traits such as soybean grain weight and grain shape at the whole genome level by association analysis, and cloned a soybean yield-regulating gene SoyZH13_17G105400 (W82 version gene number: Glyma.17G109100, also referred to as GmSW17 in this article) by integrating haplotypes, gene expression profiles, homologous gene functional annotations and previous soybean grain weight and grain shape related QTL information using molecular biology and comparative genomics. H1 、GmSW17 H2 ), providing theoretical basis and genetic resources for subsequent molecular assisted breeding and molecular design breeding.

[0008] Among them, in a specific embodiment of the present invention, the GmSW17 H1 The cDNA sequence of the gene is shown in SEQ ID NO: 1. H2 The cDNA sequence of the gene is shown in SEQ ID NO: 2. In a specific embodiment of the present invention, the GmSW17 H1 The amino acid sequence of the gene is shown in SEQ ID NO: 3. H2 The amino acid sequence of the gene is shown in SEQ ID NO: 4. In a specific embodiment of the present invention, the GmSW17 H1 The genomic DNA (gDNA) sequence of the gene is shown in SEQ ID NO: 5. H2 The genomic DNA (gDNA) sequence of the gene is shown in SEQ ID NO:6.

[0009] In the present invention, existing plant expression vectors can be used to construct recombinant vectors containing target genes. The plant expression vectors include binary Agrobacterium vectors and vectors that can be used for plant microprojectile bombardment. In order to facilitate the identification and screening of transgenic plant cells or plants, the plant expression vectors 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, antibiotic markers with resistance, or chemical agent resistance marker genes. Considering the safety of transgenic plants, no selective marker genes can be added, and transformed plants can be directly screened by adversity. The plant expression vector can also contain enhancers to increase the expression of the inserted nucleotide fragments.

[0010] To achieve the above object, the present invention also provides a method for obtaining transgenic soybeans, which comprises introducing the aforementioned nucleic acid or a vector or host cell containing the aforementioned gene into target soybeans to obtain transgenic soybeans having enlarged grains compared with the target soybeans.

[0011] Among them, the method of introducing the target soybean can be through the use of Ti plasmid, Ri plasmid, plant virus vector, direct DNA transformation, microinjection, electroporation, Agrobacterium-mediated and other conventional biological methods to transform plant cells or tissues, and cultivate the transformed soybean cells or tissues into plants.

[0012] To achieve the above object, the present invention also provides a use of the above-mentioned protein or the above-mentioned nucleic acid or a vector or host cell containing the above-mentioned gene in soybean genetic engineering.

[0013] Wherein, the soybean genetic engineering is preferably soybean genetic engineering for the purpose of increasing soybean yield.

[0014] The functions of the soybean grain weight-related protein and its encoding nucleic acid provided by the present invention in regulating soybean 100-grain weight are both discovered for the first time by the applicant, and the phenotypic analysis of transgenic plants and wild-type plants has verified that the expression of the soybean grain weight-related protein of the present invention can increase the grain weight of transgenic soybean plants. The present invention will have great theoretical and application value for soybean high-yield breeding and related application research.

[0015] Specifically, the present invention provides the following technical solutions:

[0016] In one aspect, the present invention provides an isolated gene for regulating soybean grain weight, wherein the coding region sequence of the gene is shown as SEQ ID NO: 1 or SEQ ID NO: 2 or a homologous sequence thereof.

[0017] In some embodiments, the genomic sequence of the gene is as shown in SEQ ID NO: 5 or SEQ ID NO: 6 or a homologous sequence thereof.

[0018] In another aspect, the present invention provides a protein for regulating soybean grain weight, wherein the amino acid sequence of the protein is shown in SEQ ID NO: 3 or SEQ ID NO: 4 or a homologous sequence thereof.

[0019] In another aspect, the present invention provides an expression vector comprising the gene as described above or a nucleotide sequence encoding the protein as described above.

[0020] In some embodiments, the expression vector has an antibiotic marker or a chemical resistance marker.

[0021] In another aspect, the present invention provides a host cell comprising the expression vector as described above.

[0022] On the other hand, the present invention provides a method for cultivating soybeans with increased yield, which comprises transforming the gene as described above or the nucleotide sequence encoding the protein as described above or the expression vector or host cell as described above into soybean plant cells or tissues and cultivating them to obtain soybean plants with increased yield.

[0023] In some embodiments, the vector is a plant expression vector, including a binary Agrobacterium vector and / or a vector that can be used for plant microprojectile bombardment.

[0024] In some embodiments, the host cell is selected from an E. coli cell, an Agrobacterium cell, or a plant cell.

[0025] In some embodiments, the Agrobacterium is selected from EHA105, EHA101 and GV3101

[0026] In another aspect, the present invention provides use of the gene as described above, or the nucleotide sequence encoding the protein as described above, or the expression vector or host cell as described above in cultivating soybean with improved yield.

[0027] In some embodiments, the yield improvement is manifested as an increase in grain width, an increase in 100-grain weight, and / or an increase in yield per plant.

[0028] On the other hand, the present invention provides a method for cultivating transgenic plants with increased yield, which comprises introducing the gene as described above or the nucleotide sequence encoding the protein as described above or the expression vector or host cell as described above into the target plant cell or tissue to obtain a transgenic plant, wherein the yield of the transgenic plant is increased compared with the target plant, and the plant is a leguminous plant, preferably soybean.

[0029] definition

[0030] Haplotype: It is the abbreviation of haploid genotype. In genetics, it refers to the combination of alleles at multiple loci that are inherited together on the same chromosome. In layman's terms, it is a genotype composed of several tightly linked genes that determine the same trait. It also refers to a type of single nucleotide polymorphism with statistical correlation in a chromatid. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 SoyZH13_17G105400 (W82 version gene number: Glyma.17G109100, also known as GmSW17 H1 、GmSW17 H2 ) gene cloning. Among them, a: Whole gene association analysis of soybean grain width trait. b: QQ dot plot of grain width trait. c: Association analysis display of grain width on chromosome 17. d: Schematic diagram of size distribution of all genes in the candidate interval and linkage analysis of all SNPs in the candidate interval. e: Expression analysis of all genes in the candidate interval. f: Schematic diagram of the gene structure of GmSW17. g: Two main haplotype structures of GmSW17. The significance analysis used Student's t test, *** represents P < 0.001.

[0032] Figure 2 SoyZH13_17G105400 (W82 version gene number: Glyma.17G109100, also known as GmSW17 H1 、GmSW17 H2 ) Schematic diagram of specific CRISPR-Cas9 target design for the GmSW17 gene. Among them, a: GmSW17 specific target design, the uppercase letter sequence represents the target sequence; bd: different editing single strain events of GmSW17. Among them, b is GmSW17 knockout line 1, with 1 base inserted; c is GmSW17 knockout line 2, with 2 bases missing; d is GmSW17 knockout line 3, with 6 bases missing.

[0033] Figure 3 SoyZH13_17G105400 (W82 version gene number: Glyma.17G109100, also known as GmSW17 H1 、GmSW17 H2 ) gene knockout mutants and wild-type WT (DN50). Among them, a: Comparison of grain phenotypes of transgenic plants obtained from wild-type DN50 and GmSW17 knockout lines. bd: Statistical comparison of grain width (b), 100-grain weight (c) and single-plant yield (d) of transgenic plants obtained from DN50 wild-type and GmSW17 knockout lines.

[0034] Figure 4 SoyZH13_17G105400 (W82 version gene number: Glyma.17G109100, also known as GmSW17 H1 、GmSW17 H2 )Gene transformation vector p-seed::GmSW17 H1 Schematic diagram of the spectrum.

[0035] Figure 5 Wild-type Dongnong 50 (DN50) and transfected with p-seed::GmSW17 are shown. H1 and p-seed::GmSW17 H2 Phenotypic statistical comparison of transgenic plants obtained by plasmids. Among them, a: wild-type DN50 and transfected with p-seed::GmSW17 H1 and p-seed::GmSW17 H2 Phenotypic comparison of grain size of transgenic plants obtained by plasmid. b: DN50 wild type and transfected with p-seed::GmSW17 H1 and p-seed::GmSW17 H2Detection of GmSW17 expression in transgenic plants obtained by plasmid. ce: Wild-type DN50 and transfected with p-seed::GmSW17 H1 and p-seed::GmSW17 H2 Statistical comparison of grain width (c), 100-grain weight (d) and yield per plant (e) in transgenic plants obtained by plasmid. DETAILED DESCRIPTION

[0036] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments and with reference to the accompanying drawings.

[0037] The following examples are provided to facilitate a better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples, unless otherwise specified, are conventional methods or are selected according to the product specifications. The test materials used in the following examples, unless otherwise specified, are conventional biochemical reagents that can be obtained commercially. The quantitative tests in the following examples were repeated three times, and the results were averaged.

[0038] In the following examples, the transformation recipient is Dongnong 50 (DN50), which is a variety approved by Heilongjiang Province (Heishen Bean 2007022) and can be purchased from the market. The pTF101.1 vector and Agrobacterium strain EHA101 were purchased from China Plasmid Vector Strain Cell Gene Collection Center (Biovector Science Lab, Inc).

[0039] Consumables such as enzyme digestion recovery kits were purchased from New England Biolabs and Tiangen Biochemical Technology (Beijing) Co., Ltd.

[0040] Example 1 Discovery of GmSW17 protein and its encoding gene

[0041] The inventors conducted a genome-wide association study (GWAS) using the grain width trait of 1853 soybean germplasm materials. Principal component analysis (PCA) and kinship calculation were performed using SNPs with a minimum allele frequency (MAF) ≥ 0.03. The first two components of the principal component analysis were selected to control the population structure. In addition, the Balding-Nichols (BN) matrix generated by EMMAX was used to reflect the kinship. Using the software EMMAX (Kang et al., 2010) for genome-wide association analysis, we detected a signal on chromosome 17 that was significantly associated with the grain width trait (see Figure 1a, b). The inventors analyzed the segment of chromosome 17, and combined with transcriptome data, haplotype analysis and homologous gene functional annotation, found a soybean grain weight-related protein in the segment, named GmSW17 protein (abbreviated as GmSW17), and its amino acid sequence is shown in SEQ ID NO.3 and SEQ ID NO.4. The gene encoding GmSW17 protein is named GmSW17 gene, and its cDNA sequence is shown in SEQ ID NO.1 and SEQ ID NO.2.

[0042] Example 2 Functional verification of GmSW17 protein

[0043] 1. Construction of recombinant plasmid

[0044] GmSW17 H1 and GmSW17 H2 (Glyma.17G109100)(GmSW17 H1 and GmSW17 H2 The CDS sequences of the two haplotypes of the GmSW17 gene were amplified from the cDNA of the developing seeds (R4 stage) of DN50 and Williams82, respectively. A 2Kb soybean seed-specific promoter DNA sequence, from the β-lectin α subunit encoding gene Glyma.20G148300 (Du et al., 2017), was amplified from the genomic DNA of Williams82. The connected seed-specific promoter P-seed-cDNA fragment was cloned into the pTF101.1 vector to obtain the recombinant plasmid P-seed::GmSW17 H1 and P-seed::GmSW17 H2 The specific operations are as follows:

[0045] 1. The seeds of soybean varieties DN50 and Williams82 at the R4 stage were separated from the plants, and RNA was extracted to obtain seed RNA of soybean varieties DN50 and Williams82. In addition, the leaves of soybean variety Williams82 were separated from the plants, and DNA was extracted to obtain leaf DNA of soybean variety Williams82.

[0046] 2. The total RNA obtained in step 1 was reverse transcribed into cDNA using a reverse transcription kit (Quanshijin). Using cDNA as a template, PCR amplification was performed using a primer pair consisting of F1 and R1 to obtain PCR amplification products (SEQ ID NO: 1 and SEQ ID NO: 2). Using the total DNA obtained in step 1 as a template, PCR amplification was performed using a primer pair consisting of F2 and R2 to obtain a PCR amplification product (SEQ ID NO: 7)

[0047] F1: 5'-ATGTCCTCCAAGATCAACGGTCAGA-3' (SEQ ID NO: 8);

[0048] R1: 5'-TCATGAGGCAACCTGTTTATCAGT-3' (SEQ ID NO: 9).

[0049] F2: 5'-AAGCTTGCTTGGATTTGGACCAGAC-3' (SEQ ID NO: 10);

[0050] R2: 5'-ATAGGATATTGAACTAGTTCTCG-3' (SEQ ID NO: 11).

[0051] 3. The PTF101.1 vector (purchased from China Plasmid Vector Strain Cell Gene Collection Center) was double-digested with restriction endonucleases EcoR I and Hind III to recover the vector backbone of about 9138 bp.

[0052] 4. Connect the PCR product of step 2 and the vector backbone of step 3 to obtain the recombinant plasmid P-seed::GmSW17 H1 (The diagram can be found in Figure 3 ) and P-seed::GmSW17 H2 .

[0053] 2. GmSW17 Hap1 Obtaining overexpressing transgenic plants

[0054] 1. Recombinant plasmid P-seed::GmSW17 H1 and P-seed::GmSW17 H2 The Agrobacterium strain EHA101 (purchased from China Plasmid Vector Strain Cell Gene Collection Center) was introduced to obtain recombinant Agrobacterium.

[0055] 2. The recombinant Agrobacterium obtained in step 1 was transformed into the recipient plant DN50 using the cotyledon node transformation method (see Margie et al., 2004 for the specific operation method), and T0 generation seeds were harvested. The specific operation steps are as follows:

[0056] (1) Sterilization of soybean seeds: Select full soybean seeds with no scratches on the seed coat, divide them into petri dishes, open the petri dishes and put them into a desiccator. Chlorine fumigation sterilization is used for seed disinfection. Put a small beaker in the desiccator, add 100 mL of sodium hypochlorite, then add 5 mL of concentrated hydrochloric acid, quickly cover the lid, and sterilize for 10-14 hours. After sterilization, take the soybeans out of the desiccator and blow them in a clean bench for more than 30 minutes to eliminate excess chlorine.

[0057] (2) Soybean seed imbibition: Place the sterilized seeds with the hilum facing downward into the germination medium (Table 1) and place them in a dark environment at 23°C for 16-18 hours;

[0058] (3) Preparation of Agrobacterium: Take out the bacteria stored at -80°C, add them to 5 mL of YEB medium containing the corresponding resistance (Table 2), and culture them at 28°C and 250 rpm for 24 h to revive them. Then, take 200 μL of the revived bacterial solution and add it to 200 mL of YEB medium containing the corresponding resistance, and culture them at 28°C and 250 rpm until the OD reaches 0. 600 =0.6 Collect the bacterial solution. Centrifuge at 22°C, 5,000 rpm for 10 min, and then resuspend the bacteria in liquid infection medium (i.e., infection solution) (Table 3) to an OD of 600 =0.5, 22°C, 70rpm, culture for 0.5h for later use;

[0059] (4) Preparation of explants: The imbibed seed coats were removed, and the hypocotyls away from the cotyledons were cut off with a scalpel, leaving a remaining hypocotyl of about 2-3 mm. The soybean seeds were then split into two, and the embryos were removed with a small iron brush to prepare the explants.

[0060] (5) Infection of explants: Place the prepared explants into the Agrobacterium suspension, make sure the suspension covers the explants, shake gently, and incubate for 0.5 h;

[0061] (6) Co-cultivation of explants with Agrobacterium: The infected explants were blotted on a sterile filter paper to remove excess bacterial solution, and a sterile filter paper was placed on the co-cultivation medium (Table 4). The blotted explants were placed on the co-cultivation medium and cultured at 23°C in the dark for 3-5 days.

[0062] (7) Callus induction culture: After the explants were co-cultured with Agrobacterium, the explants were transferred to callus induction medium (Table 5). At this time, the explants were tilted at 45 degrees and the bottom was gently inserted into the culture medium. The culture was carried out at 23°C, 16 h light / 8 h dark for 14 days.

[0063] (8) Bud induction culture: The induced callus was cut from the explant and placed in a bud induction medium (Table 6) at 23°C, 16 h light / 8 h dark, for 14 days, and transferred to a new bud induction medium every 14 days;

[0064] (9) Bud elongation culture: The explants with induced new buds were transferred to the bud elongation medium (Table 7), and transferred to a new bud elongation medium every 2 weeks until the buds elongated to more than 3 cm;

[0065] (10) Rooting culture: The elongated shoots were cut off and inserted into the rooting medium (Table 8) until the root length exceeded 3 cm. The seedlings were then trained and transplanted to a greenhouse at 24°C with 16 h light / 8 h dark until maturity. During this period, Basta was sprayed and positive strains were identified by PCR.

[0066] Table 1 Germination medium

[0067] GM (Germination Medium) 1L B5 (macro, micro, iron salt) 3.1g Sucrose 20g pH 5.8 Phytagel 2.5g Sterilization 121℃,20min Vitamin B5 (1000×) 1mL

[0068] Table 2 YEB medium

[0069]

[0070]

[0071] Table 3 Infection solution

[0072] LCCM (Contamination Medium) 1L B5 (macro, micro, iron salt) 0.31g Sucrose 30g MES 3.9g pH 5.4 Sterilization 121℃,20min Vitamin B5 (1000×) 1mL GA3 (1 mg / mL) 0.25mL 6-BA (1 mg / mL) 1.67mL DTT (100 mg / mL) 1.8mL As(100mg / mL) 0.8mL Silwet 20μL / 100mL

[0073] Table 4 Co-culture medium

[0074]

[0075]

[0076] Table 5 Callus induction medium

[0077]

[0078] Table 6 Bud induction medium

[0079]

[0080]

[0081] Table 7 Bud elongation medium

[0082]

[0083] Table 8 Rooting medium

[0084]

[0085] Example 3 Statistics of transgenic phenotypes of GmSW17

[0086] This example verifies that the recombinant plasmid P-seed::GmSW17 was successfully transferred into DN50. H1 and P-seed::GmSW17 H2 Afterwards, the grain width, 100-grain weight and single-plant yield of the transgenic plants obtained were significantly increased ( Figure 5), specifically, grain width increased by 6.3%, 100-grain weight increased by 8.4%, and single-plant yield increased by 15.14%.

[0087] Specifically, the transgenic strains were obtained according to the steps shown in Example 2 and cultured conventionally. The measurement results of the transgenic strains are shown in Figure 3 and Figure 5 As shown. Among them, Figure 5 -1, -2, and -3 refer to the names of transgenic lines, that is, a total of three transgenic lines (also called transgenic lines) were obtained, among which GmSW17 H1 -OE means using p-seed::GmSW17 H1 Transgenic plants obtained by plasmid transfection, DN50 refers to the wild-type DN50 plant; Figure 5 b is the expression level of the GmSW17 gene in the transgenic plants, indicating that the expression level of GmSW17 in the transgenic lines increased (among which, the soybean variety Dongnong 50 (DN50) is the wild type, that is, the non-transgenic body); Figure 5 c indicates that seed thickness increased significantly by 6.3%; Figure 5 d indicates that the 100-seed weight of the transgenic line increased significantly by 8.4% relative to DN50; Figure 5 e indicates that the seed weight per plant of the transgenic line increased significantly by 15.14% relative to DN50.

[0088] The above results show that the GmSW17 gene is a key gene regulating soybean yield. Overexpression of the GmSW17 gene in soybean plants can promote the increase of soybean 100-grain weight and grain width, thereby increasing soybean yield.

[0089] References

[0090] Du,J.,Wang,S.,He,C.,Zhou,B.,Ruan,YL,and Shou,H.(2017).Identification of regulatory networks and hub genes controlling soybean seedset and size using RNA sequencing analysis.J Exp Bot 68,1955-1972.

[0091] Du, L., Li, N., Chen, L., Xu, Y., Li, Y., Zhang, Y., Li, C., and Li, Y. (2014).Theubiquitin receptor DA1 regulates seed and organ size by modulating thestability of the ubiquitin-specific protease UBP15 / SOD2 in Arabidopsis.PlantCell 26,665-677.

[0092] Haahr, P., Borgermann, N., Guo, X., Typas, D., Achuthankutty, D., Hoffmann, S., Shearer, R., Sixma, TK, and Mailand, N. (2018).ZUFSP deubiquitylates K63-Linked polyubiquitin chains to promote genome stability.Mol Cell 70.165–174e166.

[0093] Kang, H., Sul, J., Service, S., Zaitlen, N., Kong, S., Freimer, N., Sabatti, C., and Eskin, E. (2010).

[0094] Nat Rev Mol Cell Biol 10,550-563 Commander, D., Clague, MJ, and Urbe, S. (2009).

[0095] Margie M.P.et al 2004Assessment of conditions affectingAgrobacterium-mediated soybean transformation using the cotyledonary nodeexplant.Euphytica 136:167–179.

[0096] Reyes-Turcu,F.E.,Ventii,K.H.,and Wilkinson,K.D.(2009).Regulation andcellular roles of ubiquitin-specific deubiquitinating enzymes.Annu RevBiochem 78,363-397.

[0097] Wang,S.,Wu,K.,Qian,Q.,Liu,Q.,Li,Q.,Pan,Y.,Ye,Y.,Liu,X.,Wang,J.,Zhang,J.,et al.(2017).Non-canonical regulation of SPL transcription factors by ahuman OTUB1-like deubiquitinase defines a new plant type rice associated withhigher grain yield.Cell Res 27,1142-1156.

[0098] Wu,R.,Zheng,W.,Tan,J.,Sammer,R.,Du,L.,and Lu,C.(2019).Proteinpartners of plant ubiquitin-specific proteases(UBPs).Plant Physiol Biochem145,227-236.

[0099] Yan,N.,Doelling,JH,Falbel,TG,Durski,AM,and Vierstra,RD(2000).The ubiquitin-specific protease family from Arabidopsis AtUBP1 and 2arerequired for the resistance to the amino acid analog canavanine.Plant Physiol124,1828-1843.

[0100] Wei Dong, Deng Tanggang, Ye Mao (2019). Deubiquitinase and cell cycle regulation. Chinese Journal of Cell Biology 41(06), 1135-1143.

[0101] Sequence Listing

[0102] SEQ ID NO: 1 GmSW17-H1 1779 bp cDNA Glycine max (soybean)

[0103]

[0104] SEQ ID NO:2GmSW17-H2 1779 bp cDNA Glycine max (soybean)

[0105]

[0106] SEQ ID NO:3 Glycine max (soybean) 593aa protein GmSW17-H1

[0107] MSSKINGQISPQPCPHLAEFRRTSAKPFLALHNCLRIKPPGGRAALRRDPDEVPRCCSCGLSAPSRLYACVTCAAIFCHSSTGVGLSHAAAHAASMPPGHQIAVDVDRAELFCCACHDQVYDRDFDAAVVIAQTAASTLGGDPPPPIPSPQPENLRKKRRVDYRPWAPDLRERALIGSCSVPIAGAGTVADLSPDLPRGLRGLNNLGNTCFMNSVLQALLHTPPLRNYFLSDRHNRYFCQKKNNGDVDGALKRSGGNNGNKNARTCLACDMDAMFSSVFSGDRAPYSPAKFLYSWWQHAANLATYEQQDAHEFFISMLDGIHEKVEKVRRKPHSEGNGDCCIAHKVFSGILRSDVTCMACGFTSTTYDPCIDISLDLEPNQGGSTKITTASSNHSCNGEPDCMNSSQNCGTSTLMGCLKRFTRAERLGSDQKFFCRQCQVRQETLKQMSIRKLPLVSCFHIKRFEHSSTRKMSRKVDRYLQFPFSLDMSPYLSSTILRNRFGNRIFPFDGDEPDASDELCSEFELFAVVTHSGKLDAGHYVTYLRLSNQWYKCDDAWVTQVDENIVRAAQCYMMFYVQKMLYYKATDKQVAS*

[0108] SEQ ID NO:4 Glycine max (soybean) 593aa protein GmSW17-H2

[0109] MSSKINGQISPQPCPHLAEFRRTSAKPFLALHNCLRIKPPGGRAALRRDPDEVPRCCSCGLSAPSRLYACVTCAAIFCHSSTGVGLSHAAAHAASMPPGHQIAVDVDRAELFCCACHDQVYDRDFDAAVVIAQTAASTLGGDPPPPIPSPQPENLRKKRRVDYRPWAPDLRERALIGSCSVPIAGAGTVADLSPDLPRGLRGLNNLGNTCFMNSVLQALLHTPPLRNYFLSDRHNRYFCQKKNNGDVDGALKRSGGNNGNKNARTCLACDMDAMFSSVFSGDRAPYSPAKFLYSWWQHAANLATYEQQDAHEFFISMLDGIHEKVEKVRCKPHSEGNGDCCIAHKVFSGILRSDVTCMACGFTSTTYDPCIDISLDLEPNQGGSTKITTASSNHSCNGEPDCMNSSQNCGTSTLMGCLKRFTRAERLGSDQKFFCRQCQVRQETLKQMSIRKLPLVSCFHIKRFEHSSTRKMSRKVDRYLQFPFSLDMSPYLSSTILRNRFGNRIFPFDGDEPDASDELCSEFELFAVVTHSGKLDAGHYVTYLRLSNQWYKCDDAWVTQVDENIVRAAQCYMMFYVQKMLYYKATDKQVAS*

[0110] SEQ ID NO:5 GmSW17-H1 4846bp gDNA Glycine max (soybean)

[0111]

[0112] SEQ ID NO:6GmSW17-H2 4846 bp gDNA Glycine max (soybean)

[0113]

[0114] SEQ ID NO:7 P-seed (soybean seed-specific promoter from Glyma.20G148300) 1384 bp promoter Glycine max (soybean)

[0115]

[0116] The specific embodiments described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for cultivating soybeans with increased yield, comprising transforming an isolated gene regulating soybean grain weight into soybean plant cells or tissues and cultivating them to obtain soybean plants with increased yield; The coding region sequence of the gene regulating soybean grain weight is shown in SEQ ID NO: 1 or SEQ ID NO:

2.

2. The method according to claim 1, wherein the genomic sequence of the gene regulating soybean grain weight is shown in SEQ ID NO: 5 or SEQ ID NO:

6.

3. The method according to claim 1, wherein the gene regulating soybean grain weight encodes a protein as shown in SEQ ID NO: 3 or SEQ ID NO:

4.

4. A method for cultivating soybeans with increased yield, comprising transforming an expression vector into soybean plant cells or transforming a host cell into soybean tissue and cultivating the expression vector to obtain soybean plants with increased yield; The expression vector comprises the gene defined in claim 1 or 2 or the nucleotide sequence encoding the protein defined in claim 3; The host cell comprises the expression vector. The method according to claim 4 , wherein the expression vector has an antibiotic marker or a chemical resistance marker. The method according to claim 4 , wherein the expression vector is a plant expression vector. The method according to claim 4 , wherein the expression vector is a binary Agrobacterium vector.

8. The method of claim 4, wherein the host cell is an Agrobacterium cell.

9. The method according to claim 8, wherein the Agrobacterium is selected from EHA105, EHA101 or GV3101.

10. The method according to any one of claims 1 to 9, wherein the increase in yield is manifested as an increase in grain width, an increase in 100-grain weight and / or an increase in yield per plant.

11. Use of the gene defined in claim 1 or 2, or the nucleotide sequence encoding the protein defined in claim 3, or the expression vector defined in any one of claims 4 to 6, or the host cell defined in claim 8 or 9 in cultivating soybean with improved yield.

12. The use according to claim 11, wherein the yield increase is manifested as an increase in grain width, an increase in 100-grain weight and / or an increase in single plant yield.

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