Cloning and application of genes regulating soybean seed size

Abstract

The application provides a soybean grain weight related protein, a coding gene and application thereof, wherein the coding region sequence of the gene is shown as SEQ ID NO:1 or a homologous sequence thereof. The application also provides a method for cultivating soybean with improved yield, which comprises transforming a GmENY2 gene or a vector or a host cell containing the GmENY2 gene into a soybean plant cell or tissue and cultivating to obtain a soybean plant with improved yield. Meanwhile, the application discloses the use of the GmENY2 gene or the vector or the host cell containing the GmENY2 gene in cultivating soybean with improved yield. The application has great theoretical and application values for soybean breeding and related application research.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to cloning and application of a gene for regulating soybean seed size. BACKGROUND

[0002] Soybean (Glycine max [L]. Merr.) is one of the important oil crops and economic crops in the world, and is also the main source of high-quality protein and feed protein for human beings. Soybean is originally from China, and the soybean yield per unit in China is low, so it is urgent to increase the soybean yield per unit in China.

[0003] Soybean yield is largely dependent on seed weight, i.e. seed size (including seed length, seed width and seed thickness). However, so far, the cloning of genes for regulating soybean seed size is still limited, and the genetic basis and specific regulatory mechanism in variation of soybean germplasm resources are unclear. Therefore, it is of great significance to deeply explore the molecular genetic mechanism for regulating soybean seed size for increasing soybean yield. SUMMARY

[0004] In order to further improve the yield and quality of soybean, the purpose of the present application is to provide a soybean seed weight related protein and an encoding gene and application thereof, so as to provide a theoretical basis and gene resources for subsequent molecular assisted breeding and molecular design breeding.

[0005] In the specific embodiments of the present application, the cDNA sequence of the soybean seed weight related GmENY2 gene is shown as SEQ ID NO: 1, and in the specific embodiments of the present application, the amino acid sequence of the GmENY2 gene is shown as SEQ ID NO: 2. In the specific embodiments of the present application, the genomic DNA (gDNA) sequence of the GmENY2 gene is shown as SEQ ID NO: 3.

[0006] In the present application, a recombinant vector containing a target gene can be constructed by using an existing plant expression vector. The plant expression vector includes a binary Agrobacterium vector and a vector that can be used for plant microprojectile bombardment, etc. In order to facilitate the identification and screening of transgenic plant cells or plants, the plant expression vector used can be processed, such as adding a gene that can express an enzyme or a luminescent compound in plants to produce color change, a resistant antibiotic marker or an anti-chemical reagent marker gene, etc. In consideration of the safety of transgenic plants, no selective marker gene can be added, and the transformed plants can be directly screened under stress. The plant expression vector can also contain an enhancer to increase the expression of the inserted nucleotide fragment.

[0007] To achieve the above object, the present application also provides a method for obtaining transgenic soybean, which comprises introducing the aforementioned nucleic acid or the vector or host cell comprising the aforementioned gene into the soybean of interest to obtain transgenic soybean showing increased grain size compared with the soybean of interest.

[0008] The method for introducing into the soybean of interest can be achieved by using Ti plasmid, Ri plasmid, plant virus vector, direct DNA transformation, microinjection, electroporation, Agrobacterium-mediated transformation, etc.

[0009] To achieve the above object, the present application also provides the use of the aforementioned protein or the aforementioned nucleic acid or the vector or host cell comprising the aforementioned gene in soybean genetic engineering.

[0010] The soybean genetic engineering is preferably soybean genetic engineering aiming at increasing soybean yield.

[0011] The soybean grain weight-related protein and the encoding nucleic acid thereof provided by the present application are the first discovery of the applicant in terms of the function in regulating soybean grain weight, and the phenotype analysis of transgenic plants and wild-type plants verifies that the expression of the soybean grain weight-related protein of the present application can increase the grain weight of the transgenic soybean plants. The present application has great theoretical and application value for the breeding of high-yield soybean and related application research.

[0012] Specifically, the present application provides the following technical solutions:

[0013] In one aspect, the present application 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 a homologous sequence thereof.

[0014] In some embodiments, the genomic sequence (gDNA) of the gene is shown as SEQ ID NO: 3 or a homologous sequence thereof.

[0015] In another aspect, the present application provides a protein for regulating soybean grain weight, wherein the amino acid sequence of the protein is shown as SEQ ID NO: 2 or a homologous sequence thereof.

[0016] In another aspect, the present application provides an expression vector comprising the aforementioned gene or the nucleotide sequence encoding the aforementioned protein.

[0017] In some embodiments, the expression vector has an antibiotic marker or an anti-chemical reagent marker.

[0018] In some embodiments, the antibiotic marker is selected from ampicillin, chloramphenicol, kanamycin, neomycin, rifampicin, spectinomycin, hygromycin, streptomycin and tetracycline, and the anti-chemical reagent marker is, for example, an anti-herbicide marker.

[0019] In another aspect, the present application provides a host cell comprising the expression vector as described above.

[0020] In another aspect, the present application provides a method for breeding soybean with improved yield, comprising transforming the gene as described above or the nucleotide sequence encoding the protein as described above or the expression vector as described above or the host cell as described above into a soybean plant cell or tissue and breeding to obtain a soybean plant with improved yield.

[0021] In some embodiments, the vector is a plant expression vector, including binary Agrobacterium vector and / or a vector that can be used for plant microprojectile bombardment.

[0022] In some embodiments, the host cell is selected from the group consisting of E. coli cell, Agrobacterium cell or plant cell.

[0023] In some embodiments, the Agrobacterium is selected from the group consisting of EHA105, EHA101 and GV3101

[0024] In another aspect, the present application provides use of the gene as described above or the nucleotide sequence encoding the protein as described above or the expression vector as described above or the host cell as described above in breeding soybean with improved yield.

[0025] In some embodiments, the improved yield is manifested as increased grain width, increased grain length, increased grain thickness, larger grain, increased hundred-grain weight and / or increased yield per plant.

[0026] In another aspect, the present application provides a method for breeding transgenic plants with improved yield, comprising introducing the gene as described above or the nucleotide sequence encoding the protein as described above or the expression vector as described above or the host cell as described above into a plant cell or tissue of interest to obtain a transgenic plant, wherein the yield of the transgenic plant is improved as compared to the plant of interest, and the plant is a legume, preferably soybean. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 The results of expression analysis of wild type and overexpression transgenic plants in GmENY2 (W82 version gene number: Glyma.11G168600) are shown, wherein the vertical coordinate shows the expression of GmENY2 and the horizontal coordinate shows the line number. The significance analysis uses Student's t test (*p < 0.05, ***p < 0.001).

[0028] Figure 2Phenotype statistics of wild type and transgenic plants overexpressing GmENY2 are shown. Among them, A: comparison of grain width phenotype of wild type and transgenic plants overexpressing GmENY2, the scale is 1 cm. B: comparison of grain length phenotype of wild type and transgenic plants overexpressing GmENY2, the scale is 1 cm. C: comparison of grain thickness phenotype of wild type and transgenic plants overexpressing GmENY2, the scale is 1 cm. D: statistics of grain width of wild type and transgenic plants overexpressing GmENY2. E: statistics of grain length of wild type and transgenic plants overexpressing GmENY2. F: statistics of grain thickness of wild type and transgenic plants overexpressing GmENY2. G: statistics of 100-grain weight of wild type and transgenic plants overexpressing GmENY2. H: statistics of yield per plant of wild type and transgenic plants overexpressing GmENY2. Significance analysis uses Student's t test (*p < 0.05, ***p < 0.001). DETAILED DESCRIPTION

[0029] In order to make the objects, technical solutions and advantages of the present application clearer, the present application will be further described in detail below with reference to specific embodiments and drawings.

[0030] The following examples are intended to better illustrate the present application but not to limit the present application. In the following examples, the experimental methods are conventional methods or selected according to the instructions of the commercial products unless otherwise specified. In the following examples, the experimental materials are conventional biochemical reagents available on the market unless otherwise specified. In the following examples, the quantitative tests are set up with three repeated experiments, and the results are averaged.

[0031] In the following examples, the transformation receptor is DN50, i.e. wild type, which is a Heilongjiang Province approved variety (Hei approved beans 2007022) and can be purchased on the market. Agrobacterium strain EHA101 is purchased from China Plasmid Vector Strain Cell Gene Preservation Center (Biovector Science Lab, Inc).

[0032] Consumables such as enzyme digestion recovery kit are purchased from New England Biolabs and Tian Gen Biochemical Technology (Beijing) Co., Ltd.

[0033] PFGC5941 vector was purchased from Beijing Huabodey Biotechnology Co., Ltd., item number vt-3034, vector details and use method please refer to product manual, reference literature is: Kerschen A, Napoli C A, Jorgensen R A, et al. Effectiveness of RNA interference in transgenic plants [J]. FEBS letters, 2004, 566(1-3): 223-228.

[0034] Example 1 Discovery of GmENY2 protein and its encoding gene

[0035] On the basis of a large number of sequence analysis and function verification, a protein was found from soybean variety Williams 82, which was named as GmENY2 protein, its amino acid sequence is shown as SEQ ID NO: 2 in the sequence listing, the gene encoding GmENY2 protein was named as GmENY2 gene, its genomic sequence is shown as SEQ ID NO: 3 in the sequence listing, and its cDNA sequence is shown as SEQ ID NO: 1 in the sequence listing.

[0036] Example 2 Function verification of GmENY2 protein

[0037] I. Construction of recombinant plasmid

[0038] 1. The seeds of soybean variety Williams 82 R4 stage (full pod stage) were separated from the plants, and RNA was extracted to obtain the seed RNA of soybean variety Williams 82.

[0039] 2. The total RNA obtained in step 1 was reverse transcribed into cDNA using a reverse transcription kit (Quangen). The cDNA was used as a template for PCR amplification with a primer pair composed of F1 and R1 to obtain a PCR amplification product.

[0040] F1: 5'-ATGAAGCCGAAGGCGTCAGTGAA-3' (SEQ ID NO: 4);

[0041] R1: 5'-AAGTGCAGCAGACACAAGAAACG-3' (SEQ ID NO: 5).

[0042] 3. The PFGC5941 vector was double digested with restriction endonuclease AscI and XmaI, and a vector backbone of about 9992 bp was recovered.

[0043] 4. The PCR product of step 2 was amplified with a primer pair composed of F2 and R2 to obtain PCR amplification product 2.

[0044] F2: 5'-cattacaattacatttacaattaATGAAGCCGAAGGCGTCAGTGAA-3' (SEQ ID NO: 6);

[0045] R2: 5'-caggactctagggactagtcccgggTCAAGCGTAATCTGGAACGTC-3' (SEQ ID NO: 7).

[0046] 5, The PCR product of step 4 and the vector skeleton of step 3 are connected to obtain a recombinant plasmid. According to the sequencing result, the structure of the recombinant plasmid A is described as follows: a double-stranded DNA molecule as shown in SEQ ID NO: 1 is inserted between the AscI I and Xma I enzyme cutting sites of the PFGC5941 vector.

[0047] II. Obtaining of GmENY2 overexpression transgenic plants

[0048] 1, The recombinant plasmid is introduced into Agrobacterium strain EHA101 (purchased from China Plasmid Vector Strain Cell Gene Preservation Center) to obtain a recombinant Agrobacterium.

[0049] 2, The recombinant Agrobacterium obtained in step 1 is used to transform the receptor plant DN50 by the cotyledon node transformation method (for specific operation method, see Margie M. P. et al 2004 Assessment of conditions affecting Agrobacterium-mediated soybean transformation using the cotyledonary node explant. Euphytica 136: 167-179), and the T0 generation seeds are harvested. The specific operation steps are as follows:

[0050] (1) Sterilization of soybean seeds: select full soybean seeds without scratches on the seed coat, divide and place them in a culture dish, place the culture dish open in a desiccator, and sterilize the seeds by chlorine fumigation. A small beaker is placed in the desiccator, 100 mL of sodium hypochlorite is added, then 5 mL of concentrated hydrochloric acid is added, the lid is quickly closed, and sterilization is performed for 10-14 h. After sterilization is completed, the soybeans are taken out of the desiccator and blown for more than 30 min in a clean bench to eliminate excess chlorine;

[0051] (2) Soybean seed imbibition: insert the sterilized seed nodule into the germination medium (Table 1) with the seed nodule facing down, place it in a 23°C dark environment for 16-18 h;

[0052] (3) Agrobacterium preparation: Agrobacterium stored at -80°C was taken out and added to 5 mL of YEB medium (Table 2) containing the corresponding resistance, and cultured at 28°C, 250 rpm for 24 h to recover, then 200 μL of the recovered bacterial solution was taken and added to 200 mL of YEB medium containing the corresponding resistance, and cultured at 28°C, 250 rpm until the OD 600 = 0.6. The bacterial solution was collected. Centrifugation was performed at 22°C, 5,000 rpm for 10 min, and then the bacterial solution was resuspended in liquid infiltration medium (Table 3) to an OD 600 = 0.5, and cultured at 22°C, 70 rpm for 0.5 h for standby use;

[0053] (4) Preparation of explants: The swollen seed coat was taken out, the hypocotyl was cut away from the cotyledon with a scalpel, and the remaining hypocotyl was about 2-3 mm, then the soybean seed was cut into two, and the embryo was removed with a small iron brush to obtain the prepared explants;

[0054] (5) Infection of explants: The prepared explants were placed in the Agrobacterium suspension, and the resuspension was covered with the explants, and gently shaken for 0.5 h;

[0055] (6) Co-culture of explants and Agrobacterium: The infected explants were blotted on sterile filter paper to remove excess bacterial solution, and a sterile filter paper was placed on the co-culture medium (Table 4), and the blotted explants were placed on the co-culture medium, and cultured at 23°C in the dark for 3-5 d;

[0056] (7) Callus induction culture: After the co-culture of the explants and Agrobacterium was completed, the explants were transferred to the callus induction medium (Table 5), at this time the explants were inserted into the medium at an angle of 45 degrees, and the bottom was gently inserted into the medium, and cultured at 23°C, 16 h light / 8 h dark for 14 d;

[0057] (8) Bud induction culture: The induced callus was cut from the explants and placed in the bud induction medium (Table 6), and cultured at 23°C, 16 h light / 8 h dark for 14 d, and transferred to new bud induction medium every 14 d;

[0058] (9) Bud elongation culture: The explants with induced new buds were transferred to the bud elongation medium (Table 7), and transferred to new bud elongation medium every 2 weeks until the buds elongated to more than 3 cm;

[0059] (10) Rooting culture: The elongated buds were cut and inserted into the rooting medium (Table 8) until the roots were longer than 3 cm, then the seedlings were hardened and transplanted to the greenhouse, and cultured at 24°C, 16 h light / 8 h dark until maturity, during which Basta and PCR identification were performed on the positive strains.

[0060] Table 1 Germination medium

[0061] GM (germination medium) 1L B5 (macro, micro, iron salts) 3.1g Sucrose 20g pH 5.8 Phytagel 2.5g Sterilization 121 °C, 20 min B5 vitamins (1000x) 1 mL

[0062] Table 2 YEB medium

[0063]

[0064]

[0065] Table 3 Infection solution

[0066]

[0067] Table 4 Co-culture medium

[0068]

[0069]

[0070] Table 5 Callus induction medium

[0071]

[0072] Table 6 Shoot induction medium

[0073]

[0074]

[0075] Table 7 Shoot elongation medium

[0076]

[0077] Table 8 Rooting medium

[0078]

[0079]

[0080] Example 3 Transgenic phenotype statistics of GmENY2

[0081] 1. Identification of expression amount of gene

[0082] The following identifications were made on the recipient line DN50 and the GmENY2 overexpression transgenic line, respectively:

[0083] (1) DN50 and GmENY2 overexpression transgenic R4 stage seeds were separated from the plants, total RNA was extracted and reverse transcribed into cDNA.

[0084] (2) The cDNA extracted in step (1) was used as a template to identify the expression amount of GmENY2 gene using primers composed of F4 and R4, and to identify the expression amount of the internal reference gene (Actin gene) using F3 and R3.

[0085] F3: 5'-CGGTGGTTCTATCTTGGCATC-3' (SEQ ID NO: 8);

[0086] R3: 5'-GTCTTTCGCTTCAATAACCCTA-3' (SEQ ID NO: 9).

[0087] F4: 5'-ACTCTCTGCAGAGCCGTTGTGAA-3' (SEQ ID NO: 10);

[0088] R4: 5'-AAGTGCAGCAGACACAAGA-3' (SEQ ID NO: 11).

[0089] The expression of GmENY2 gene in different materials was obtained by quantitative PCR amplification with each specific primer pair using the cDNA as a template, and the results are shown in Table 1. Figure 1 As can be seen from Table 1, the expression of GmENY2 gene in the overexpression lines GmENY2-OE-1 and GmENY2-OE-2 was significantly higher than that in the control DN50. Figure 1

[0090] 2. Comparison of grain size, grain weight and yield per plant of the recipient line DN50 and the GmENY2 overexpression transgenic lines

[0091] We counted the grain size, hundred-grain weight and yield per plant of the seeds of the recipient line DN50 and the GmENY2 overexpression transgenic lines, and the results are shown in Table 2. Figure 2 As can be seen from Table 2, the grains of the GmENY2 overexpression transgenic lines were significantly larger than those of DN50 (specifically, the grain width increased by 5.6%, the grain length increased by 1.6%, and the grain thickness increased by 5.7%), which indicated that GmENY2 gene was a key gene for regulating soybean yield, and overexpression of GmENY2 gene in soybean plants could promote the soybean grains to become larger, increase the hundred-grain weight (about 7.2%), and thus increase the yield per plant (by 20%). Figure 2 SEQUENCE LISTING

[0092] SEQ ID NO: 1 GmENY2 345bp cDNA Glycine max (soybean)

[0093]

[0094] ​​ATGAAGCCGAAGGCGTCAGTGAATCGACCCCCCACACCAGATGTGGCCGAAAATGCCCCTGAAAGGGAACCCACGCTTCAAGAGCTCATCAACATCAAGTTGATCGAGACCGGAGAGAAGGAGCGTCTCATGGAGCTGTTGAGGGAAAGGCTTGTTGATTGCGGTTGGAAGGATGAAATGAAAACTCTCTGCAGAGCCGTTGTGAAGAAGAAAGGGAGGAATAATGTTACTGTTGATGAACTTATACATGTAATCACTCCAAAGGGACGAGCCTCGATTCCTGATTCCGTAAAGGCCGAGTTGTTGCAGAGGATTCAAACGTTTCTTGTGTCTGCTGCACTTTAA

[0095] SEQ ID NO: 2 GmENY2 115 aa protein Glycine max (soybean)

[0096] MKPKASVNRPPTPDVAENAPEREPTLQELINIKLIETGEKERLMELLRERLVDCGWKDEMKT LCRAVVKKKGRNNVTVDELIHVITPKGRASIPDSVKAELLQRIQTFLVSAAL*

[0097] SEQ ID NO: 3 GmENY2 2722 bp gDNA Glycine max (soybean)

[0098]

[0099] The above-described specific embodiments further illustrate the objects, technical solutions and advantages of the present application. It should be understood that the above-described specific embodiments are merely for the purpose of illustrating the present application, and are not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method for cultivating soybeans with increased yield, characterized in that, The method includes transforming a gene that regulates soybean grain weight into soybean plant cells or tissues and cultivating them to obtain soybean plants with increased yield. The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO:

2.

2. The method according to claim 1, characterized in that, The coding region sequence of the gene is shown in SEQ ID NO:

1.

3. A method for cultivating soybeans with increased yield, characterized in that, The method includes transforming a gene that regulates soybean grain weight into soybean plant cells or tissues and cultivating them to obtain soybean plants with increased yield. The genomic sequence of the gene is shown in SEQ ID NO:

3.

4. A method for cultivating soybeans with increased yield, characterized in that, The method includes transforming an expression vector containing a gene that regulates soybean grain weight into soybean plant cells or tissues and culturing them to obtain soybean plants with increased yield. The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO:

2.

5. The method according to claim 4, characterized in that, The expression vector is labeled with antibiotics.

6. The method according to claim 5, characterized in that, The antibiotic labels are selected from ampicillin labels, chloramphenicol labels, kanamycin labels, neomycin labels, rifampin labels, spectinomycin labels, hygromycin labels, streptomycin labels, and tetracycline labels.

7. The method according to claim 4, characterized in that, The expression vector is labeled with resistance to chemical reagents.

8. The method according to claim 7, characterized in that, The chemical resistance marker is a herbicide resistance marker.

9. The method according to claim 4, characterized in that, The vector is a binary Agrobacterium plant expression vector.

10. The method according to claim 4, characterized in that, The vector is a plant expression vector that can be used for plant micro-bombardment.

11. A method for cultivating soybeans with increased yield, characterized in that, The method includes transforming a host cell containing an expression vector as defined in any one of claims 4 to 10 into soybean plant cells or tissues and culturing them to obtain soybean plants with increased yield, wherein the host cell is Agrobacterium cells.

12. The method according to claim 11, characterized in that, The Agrobacterium was selected from EHA105, EHA101 and GV3101.

13. The method according to any one of claims 1 to 12, characterized in that, The increased yield is manifested in increased grain width, increased grain length, increased grain thickness, larger grains, increased weight per 100 grains, and / or increased yield per plant.

14. Use of the gene as defined in any one of claims 1 to 3, or the expression vector as defined in any one of claims 4 to 10, or the host cell as defined in any one of claims 11 to 12, in the cultivation of soybeans with increased yield.

15. The use according to claim 14, characterized in that, The increased yield is manifested in increased grain width, increased grain length, increased grain thickness, larger grains, increased weight per 100 grains, and / or increased yield per plant.

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