Soybean GmHRF-1 gene and application of protein coded by soybean GmHRF-1 gene in improvement of soybean regeneration frequency and genetic transformation efficiency

Abstract

The invention discloses a soybean GmHRF-1 gene and application of protein coded by the soybean GmHRF-1 gene in improvement of soybean regeneration frequency and genetic transformation efficiency, and relates to the technical field of plant genetic engineering. The invention identifies and verifies that the GmHRF-1 gene in the soybean B3 transcription factor family has a brand new function of remarkably improving the regeneration capacity of somatic cells for the first time. By introducing the gene into soybean recipient cells and overexpressing the GmHRF-1, the regeneration frequency and genetic transformation efficiency of soybeans can be remarkably improved. The invention provides an efficient and universal endogenous gene for breaking through the technical bottleneck of genetic transformation of backbone germplasm which is difficult to regenerate of soybeans. The research result not only provides a new gene target for analyzing a molecular mechanism of soybean stem tip meristem regulation and regeneration, but also provides a technical support for constructing an efficient and stable soybean genetic transformation system.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of plant genetic engineering, and particularly relates to a soybean GmHRF-1 gene and the protein coded by the gene in improving the regeneration frequency and genetic transformation efficiency of soybean. BACKGROUND

[0002] Soybean is one of the most important food and economic crops in the world. However, soybean is a recognized difficult-to-transfect crop, and the success of soybean genetic transformation depends on two core links: one is the efficient introduction of exogenous genes into soybean recipient cells, and the other is the regeneration of transformed cells into whole plants through tissue culture. Previous studies have shown that the regeneration frequency of transformed cells and the transformation efficiency of explants are largely dependent on the adaptability of the recipient system (such as explant type, genotype) and the transformation method. Therefore, improving the regeneration ability of explant cells and optimizing the transformation efficiency are the key to breaking through the technical bottleneck of soybean genetic transformation and promoting the large-scale application of genetic engineering breeding.

[0003] In this study, based on the previous multi-omics data joint analysis of the research group, a candidate gene GmHRF-1 that affects the regeneration ability and genetic transformation efficiency of soybean was screened. The gene belongs to the soybean B3 (B3 transcription factor) transcription factor family. The B3 transcription factor family is a large plant-specific transcription factor family, which can be divided into multiple subfamilies according to the domain characteristics, such as ARF (Auxin Response Factor), LEC2 (LEAFY COTYLEDON 2), VAL (VP1 / ABI3-like) and REM (Reproductive Meristem) etc. A large number of studies have revealed that members of this family have various important functions in plant development: in the model plant Arabidopsis thaliana, the ARF subfamily mainly regulates auxin response; members such as LEC2 and FUS3 are core regulators of embryonic development and seed maturation; VAL1 / VAL2 is involved in meristem maintenance and differentiation. At present, the research on the B3 family in soybean is relatively lagging behind, and most of the research is focused on the ARF subfamily, focusing on its regulation of root development, stress response and yield-related traits.

[0004] At present, whether it is the model plant Arabidopsis or soybean, the research on B3 family has long focused on embryo development, hormone response and specific organ building, and there is no clear evidence to show that members of this family can directly and significantly regulate the regeneration ability of soybean somatic cells (such as cotyledon nodes). In addition, due to the great functional differentiation of different species and different subfamily members, it is itself a work full of uncertainties to find and verify the B3 gene that can specifically improve the regeneration efficiency of soybean. It is a key problem to be solved in the field to explore the gene target that can directly and efficiently regulate the in vitro regeneration ability of soybean from the genetic background of soybean itself. SUMMARY

[0005] In view of the above prior art, the purpose of the present application is to provide a soybean GmHRF-1 gene and the protein encoded by the gene in improving the regeneration frequency and genetic transformation efficiency of soybean. The present application research found that GmHRF-1 is a soybean regeneration related gene, and overexpression of GmHRF-1 gene can significantly improve the regeneration frequency and genetic transformation efficiency of soybean. The present research can provide an important reference for the creation of new transgenic soybean germplasm and molecular breeding application.

[0006] To achieve the above purpose, the technical scheme adopted by the present application is as follows: In the first aspect of the present application, the application of soybean GmHRF-1 gene in the following (1) or (2) is provided: (1) improving the regeneration frequency of soybean; (2) improving the genetic transformation efficiency of soybean; The soybean GmHRF-1 is a DNA molecule as shown in i) or ii) or iii) below: i) the nucleotide sequence is a DNA molecule as shown in SEQ ID NO. 1, specifically as follows: SEQ ID NO. 1:

[0007] ii) a DNA molecule encoding the amino acid sequence shown in SEQ ID NO. 2 other than that in i); iii) a DNA molecule having 90% or more identity with the DNA fragment defined in i) or ii), and encoding a protein functionally equivalent to the protein shown in SEQ ID NO. 2.

[0008] The term "increasing the regeneration frequency of soybean" refers to a significant increase in the percentage of explants (such as cotyledon nodes) that can successfully initiate and complete the regeneration process during the tissue culture (in vitro culture) of soybean.

[0009] Preferably, the increasing the regeneration frequency of soybean refers to increasing the regeneration efficiency of soybean cotyledon nodes.

[0010] Preferably, the increasing the regeneration frequency of soybean and / or increasing the genetic transformation efficiency of soybean is achieved by promoting the expression of a soybean GmHRF-1 gene.

[0011] Preferably, the substance that promotes the expression of a soybean GmHRF-1 gene is any one of the following: C1) an expression cassette containing GmHRF-1 the gene; C2) a recombinant vector containing GmHRF-1 the gene, or a recombinant vector containing the expression cassette of C1); C3) a recombinant microorganism containing GmHRF-1 the gene, or a recombinant microorganism containing the expression cassette of C1), or a recombinant microorganism containing the recombinant vector of C2); C4) a transgenic plant cell line containing GmHRF-1 the gene, or a transgenic plant cell line containing the expression cassette of C1).

[0012] In a second aspect of the present application, a method for increasing the regeneration frequency of soybean and / or increasing the genetic transformation efficiency of soybean is provided, comprising the following steps: introducing a soybean GmHRF-1 gene into an expression vector to construct a recombinant expression vector, transforming the recombinant expression vector into an Agrobacterium competent cell to obtain an Agrobacterium strain for transformation, and using the Agrobacterium strain to infect soybean cotyledons to obtain a transgenic soybean plant with increased regeneration efficiency.

[0013] Preferably, the expression vector is a pPTN1171 vector, and the Agrobacterium competent cell is Agrobacterium EHA105.

[0014] In a third aspect of the present application, the above-mentioned soybean GmHRF-1The application of the protein encoded by the gene in (1) or (2) below: (1) increasing the regeneration frequency of soybean; (2) increasing the genetic transformation efficiency of soybean; The soybean GmHRF-1 The protein encoded by the gene is the protein shown in (A1) or (A2) below: (A1) a protein consisting of the amino acid sequence shown in SEQ ID NO. 2 in the sequence listing, specifically as follows: SEQ ID NO. 2: MVGQNCDGCRSWEEDIYWSHFQFLHFVQFLHADYDQHLALPKTFSDNLKKKLPENVTLKGPGGVMWNIGMTTRDDTLYFGHGWEQFVKDHCLKENDFLVFKYNGESQFDVLIFNGWSLCEKAGSYFVRKCGHTEIDHAGGSLNKKRDTDNDSLEEGNIPSNAGVECALHEKSAHVNGTKEPIDVPPETPPTENTFNAGVESSGVEQFTPDGGVTLAAVPSETANGKRIRNIVSAVKHVHTKRKGRPAKWHVRERTLDWVAALEAEPVSASRSGTYEVYKSNRRPVTDDETRKIESLAKAACTDDSIYVVMKPTHVYKRFFVSIRGTWIGKHISPSSQDVILRMGKGEWIARYSYNNIRNNGGLTGGWKHFSLDSNLEEGDACVFKPAGQINNTFVIDMSIFRVVPETVPLTPMSRGTRTGTRTGTGRRGRKPATMKSIQTQLSSP.

[0015] (A2) a fusion protein obtained by connecting a protein tag to the N terminal and / or C terminal of the protein defined in (A1).

[0016] Preferably, the regeneration frequency of soybean and / or the genetic transformation efficiency of soybean is increased by increasing the activity of the protein encoded by the gene. GmHRF-1 The protein encoded by the gene.

[0017] In the fourth aspect of the present application, the recombinant expression vector or genetically engineered bacteria containing the above-mentioned soybean GmHRF-1 The application of the recombinant expression vector or genetically engineered bacteria containing the above-mentioned soybean (1) increasing the regeneration frequency of soybean; (2) increasing the genetic transformation efficiency of soybean.

[0018] The present application has the following advantages: This invention is the first to identify and verify the presence of the soybean B3 transcription factor family. GmHRF-1 This gene possesses a novel function that significantly enhances the somatic cell regeneration capacity of soybeans. By introducing this gene into soybean recipient cells and overexpressing it... Figure 1 This study can significantly improve the regeneration frequency and genetic transformation efficiency of soybeans. It provides a highly efficient and universally applicable endogenous gene for overcoming the bottleneck in the genetic transformation technology of key soybean germplasm that is difficult to regenerate. The results not only provide new gene targets for elucidating the molecular mechanism of regeneration regulation by soybean shoot tip meristem, but also provide technical support for constructing an efficient and stable soybean genetic transformation system. Attached Figure Description

[0019] Figure 2 Amino acid sequence comparison diagram of soybean GmHRF-1 and Arabidopsis thaliana AtHRF.

[0020] Figure 3 Phylogenetic analysis of GmHRF-1 homologous proteins.

[0021] pPTN1171::GmHRF-1 : pPTN1171::GmHRF-1 Comparison of the transformation process of Williams82 with the control empty vector; in the figures, Figures A-E show the soybean cotyledonary node tissue culture process of W82 transformed with pPTN1171 empty vector, and Figures F-J show... Figure 4 Tissue culture process of soybean cotyledonary nodes transformed with W82. CCM: co-culture medium; SIM: shoot induction medium.

[0022] GmHRF-1 Overexpression Figure 5 Transform Williams82 data for statistical analysis.

[0023] GmHRF-1 : GmHRF-1 Image showing the electrophoretic identification results of transformed positive plants. Detailed Implementation

[0024] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0025] As mentioned earlier, efficient regeneration in soybeans is a crucial foundation for genetic transformation. However, due to the complex genetic composition of soybeans, as well as their tissue-specific and genotype-specific characteristics, research on the genetic mechanisms of regeneration traits faces numerous limitations. Consequently, research on soybean gene function and genetic breeding improvement lags significantly behind other staple crops such as rice and maize. Currently, there are very few reports on genes that improve the regeneration efficiency and genetic transformation efficiency of soybean cotyledonary nodes.

[0026] Therefore, the gene capable of regulating soybean cotyledon node regeneration efficiency is researched in the application. GmHRF-1 The gene has a positive regulation effect in the soybean cotyledon node regeneration process. By comparing the amino acid sequences, it is found that the protein coded by the gene has the highest sequence similarity with AtREM16 (gene ID: AT3G53310) protein sequence in Arabidopsis thaliana. In the phylogenetic tree, AtREM16 is added with HRF alias, namely (AtHRF), which is only used for the functional correlation annotation in the present research, and the original gene name and annotation are still based on the TAIR database. GmHRF-1 The nucleotide sequence is shown as SEQ ID NO. 1, the coding sequence of the gene is 1338 bp in length, the amino acid sequence is shown as SEQ ID NO. 2, and 445 amino acids are coded. At present GmHRF-1 The biological function of the gene in soybean has not been reported.

[0027] The gene is amplified from the Williams82 stem tip meristem cDNA in the application GmHRF-1 The gene is amplified from the Williams82 stem tip meristem cDNA in the application GmHRF-1 The overexpression vector is constructed by using the DNA sequence of the CDS full-length fragment of the gene, then the overexpression vector is introduced into Agrobacterium, and the Agrobacterium-mediated method is used to infect soybean, then subculture is carried out until the transgenic positive strain is obtained. In the subculture process, the inventors find that overexpression of the gene can significantly improve the regeneration frequency of soybean. GmHRF-1 Improving the regeneration efficiency is an important way to improve the genetic transformation efficiency of soybean, can lay a good foundation for transforming other excellent genes, and can produce important economic value and social benefits.

[0028] In order to enable those skilled in the art to more clearly understand the technical solutions of the present application, the technical solutions of the present application will be described in detail below in combination with specific embodiments.

[0029] The test materials not specifically described in the embodiments of the present application are all conventional test materials in the art, and can be purchased through commercial channels. In the embodiments of the present application, if the specific experimental conditions and methods are not specified, the conventional conditions are usually used, such as J. Sambrook et al., Science Press, 2002, Molecular Cloning Experiment Guide (3rd Edition); D.L. Specter et al., Science Press, 2001, Cell Experiment Guide; or the conditions recommended by the manufacturer.

[0030] Among them, the composition of the culture medium used in the embodiments of the present application is as follows: Co-culture medium (CCM): B5 powder 3.9g, sucrose 30g, MES 3.9g, 6-BA 1.67mg, distilled water to a final volume of 1L; cysteine ​​400mg, dithiothreitol 154.2mg, gibberellin 0.25mg, dimethyl sulfoxide 40mg, agar 8g, distilled water to a final volume of 1L; pH=5.4.

[0031] Shoot induction medium (SIM): B5 powder 3.9g, sucrose 30g, MES 0.59g, 6-BA 1.67mg, termethin 250mg, cefotaxime sodium 100mg, glyphosate 5mg, agar 8g, distilled water to a final volume of 1L; pH=5.7.

[0032] Shoot elongation medium (SEM): B5 powder 4.3g, sucrose 30g, MES 0.59g, asparagine 50mg, glutamine 50mg, termethin 250mg, cefotaxime sodium 100mg, zeatin 1mg, gibberellin 0.5mg, auxin 0.1mg, agar 8g, activated charcoal 2g, distilled water to a final volume of 1L; pH=5.7.

[0033] Example 1: GmHRF-1 Gene cloning and construction of plant overexpression vectors 1. Extraction and purification of total RNA from soybeans: Amplification from Williams82 shoot apical meristem GmHRF-1 Genes. The RNA extraction kit used in this experiment was the UltraPure RNA Extraction Kit provided by Beijing Kangwei Reagent Biotechnology Co., Ltd. Specific experimental procedures were performed according to the kit's instructions.

[0034] To ensure that the RNA quality met the requirements, the purity and concentration of the purified RNA samples were detected using a spectrophotometer and agarose gel electrophoresis, respectively. The purity and concentration standards were as follows: RNA purity was OD0.05. 260 / 280 and OD 260 / 230 All values ​​were in the range of 1.8-2.0, and the RNA concentration was in the range of 1.0-2.0 µg / µL.

[0035] 2. Synthesis of the first strand of cDNA: The cDNA first strand synthesis kit used in this experiment was completed using the FastQuantRT Kit (with gDNase) from Tiangen Biotech Co., Ltd.

[0036] 3. pPTN1171::GmHRF-1 Gene cloning: Using reverse-transcribed cDNA as a template, PCR amplification was performed using the following primer pairs: Upstream primer: 5'-ATGGTTGGTCAAAATTGTGA-3' (SEQ ID NO. 3); Downstream primer: 5'-TGGTGAACTAAGTTGTGTCT-3' (SEQ ID NO. 4); The PCR amplification system was 1 μL of upstream primer (10 pmol / μL), 1 μL of downstream primer (10 pmol / μL), 12.5 μL of 2x Phanta Max Master Mix, 1 μL of cDNA template, and DEPC-H2O was added to make up the total volume to 25 μL.

[0037] The amplification conditions were: 95°C pre-denaturation for 5 min; 95°C denaturation for 30 s, 60°C annealing for 30 s, 72°C extension for 1 min, 34 cycles; 72°C extension for 5 min.

[0038] 4. Construction of plant expression vector GmHRF-1 (1) 4 μL of PCR product was ligated with the digested pPTN1171 vector. (2) The ligation product was transformed into E. coli Top10 and cultured on LB solid medium containing spectinomycin (100 mg / L) overnight.

[0039] (3) A single colony was picked and cultured in LB liquid medium containing spectinomycin (100 mg / L) overnight. Plasmid DNA was extracted by alkaline method and sequenced.

[0040] (4) The sequence of the amplified product was analyzed, which was shown in SEQ ID NO. 1, indicating that

[0041] the gene had been ligated to the pPTN1171 vector, and the expression vector was constructed. Figure 3

[0042] 5. Agrobacterium-mediated transformation (1) 2 μL of overexpression vector plasmid was added to 20 μL of EHA105 competent cells, ice bath for 5 minutes, put into liquid nitrogen for 5 minutes, 37°C for 5 minutes, ice bath for 5 minutes, add 1 ml of YEP liquid medium without antibiotics, 28°C, 180 rpm shaking for 3 hours, then centrifuged and plated on YEP solid medium containing spectinomycin (100 mg / L) and rifampicin (25 mg / L), and cultured at 28°C for 2 days.

[0043] ​(2) Pick single colony and inoculate into YEP liquid medium containing spectinomycin (100 mg / L), rifampicin (25 mg / L) at 28°C, 200 rpm for 16 h. Perform bacterial liquid PCR identification using cloning primers. Positive clone bacterial liquid glycerol preservation is stored at -80°C for standby.

[0044] Example 2: Agrobacterium-mediated genetic transformation of soybean 1. Genetic transformation of soybean: The genetic transformation of soybean adopts Agrobacterium-mediated cotyledon node transformation method. The transformation process is shown in pPTN1171:: GmHRF-1 The specific transformation steps are as follows: (1) Select mature and full soybean seeds without disease spots and seed coat rupture, arrange them in a single layer in 90x15mm culture dishes, place them in a desiccator and in a fume hood overnight, and sterilize them with chlorine for 15 hours.

[0045] (2) After sterilization, place the soybean seeds in a sterile table and blow them with strong wind for 120 minutes to remove the residual chlorine in the culture dish.

[0046] (3) After sterilization, place the soybean seeds in a sterilized tissue culture bottle and add sterile water to soak. After sterilization, the soybean seeds are soaked in the dark at 20°C for 12 hours.

[0047] (4) Take 1 ml of glycerol-preserved positive bacterial liquid and add it to 5 ml of YEP liquid medium containing spectinomycin (100 mg / L), rifampicin (25 mg / L). Incubate overnight at 28°C on a shaker.

[0048] (5) Inoculate the shaken bacterial liquid into 100 ml of YEP liquid medium containing spectinomycin (100 mg / L), rifampicin (25 mg / L). Incubate at 28°C for 6 hours.

[0049] (6) Cut the seed along the seed umbilical cord with a sterilized scalpel blade to separate the cotyledon into two parts, obtaining the transgenic recipient.

[0050] (7) Separate the bacterial liquid with OD value of 0.6-1.0 into a 50 ml sterile centrifuge tube, centrifuge at 4000 rpm for 10 minutes, discard the supernatant, and add 50 ml of liquid CCM medium to resuspend the bacterial cells, and set aside.

[0051] (8) Soak the prepared soybean transgenic recipient in the bacterial liquid for 25 minutes, and frequently stir the bacterial liquid to ensure that the explants are in contact with fresh bacterial liquid.

[0052] (9) The explants were removed from the bacterial suspension and blotted dry with sterile absorbent paper, then placed on solid co-cultivation medium CCM with sterile filter paper on top. The Petri dishes were stacked, sealed with plastic wrap, and transferred to a Percival incubator (23°C) for 4 days of dark incubation.

[0053] (10) After 4 days of co-cultivation on CCM, the elongated hypocotyls were excised to about 0.3 cm and placed on shoot induction medium SIM. The explant hypocotyls were inserted into the medium with 6 explants per dish. The Petri dishes were sealed with 12 mm wide 3M breathable tape and placed in the culture room (24°C, 18 / 6 h light cycle, 140 μmol / m 2 / sec light intensity) for 30 days, with subculturing every 15 days.

[0054] (11) The differentiated explants were transferred to shoot elongation medium SEM, and those that were dormant at the end of the SIM phase and those that were dead were discarded. The cotyledons were excised from the explants, and a fresh cut was made at the base of the developing node. The explants were placed in the culture room (24°C, 18 / 6 h light cycle, 140 μmol / m 2 / sec light intensity) for 6 weeks until the shoots were rooted.

[0055] Example 3: Evaluation of Agrobacterium-mediated transformation efficiency of soybean The transformation conditions described in Example 2 were followed, and pPTN1171 empty vector was used as a control. The regeneration frequency (the ratio of explants that sprouted) and the regeneration shoot rate (the ratio of explants with shoots longer than 3 cm) of pPTN1171 empty vector and GmHRF-1 were calculated. The genomic DNA of the regenerated shoots was extracted, and PCR identification was performed. The positive shoot rate (transformation efficiency) was calculated. The formulas involved in the calculation process are as follows: Regeneration frequency = the number of regenerated shoots / the total number of explants x 100%; Regeneration shoot rate = the number of explants with shoots longer than 3 cm on the shoot elongation medium / the total number of explants x 100%; Positive shoot rate = the number of positive shoots identified by PCR (at least two reliable repetitions) / the total number of explants x 100%; Table 1: First batch of transformation data of Williams 82 Table 2: Second batch of transformation data of Williams 82 Table 3: Third batch of transformation data of Williams 82 Table 4: Statistics of regeneration frequency, regeneration shoot rate, and positive shoot rate The experimental results show that, taking the transformation of empty vector pPTN1171 as a control, the experimental groups overexpressing Figure 4-5 all showed significantly higher regeneration frequency in three batches of independent repeated experiments, and the effects of regeneration seedling rate and positive seedling rate were excellent (as shown in Tables 1-4). GmHRF-1 This shows that GmHRF-1 overexpression of the gene can effectively break the soybean regeneration restriction, greatly improve the soybean regeneration frequency and genetic transformation efficiency.

[0056] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Those skilled in the art can make various modifications and changes to the present application. Any modification equivalent replacement, improvement, etc. within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. Soybeans GmHRF-1 The application of genes in the following (1) or (2): (1) Increase the frequency of soybean regeneration; (2) Improve the efficiency of soybean genetic transformation; The soybean GmHRF-1 It is a DNA molecule as shown in i), ii), or iii): i) The nucleotide sequence is the DNA molecule shown in SEQ ID NO.1; ii) DNA molecules other than i) encoding the amino acid sequence shown in SEQ ID NO.2; iii) A DNA molecule that has 90% or more identity with the DNA fragment defined in i) or ii) and encodes a protein that is functionally equivalent to the protein shown in SEQ ID NO.

2.

2. The application according to claim 1, characterized in that, The increase in soybean regeneration frequency refers to the increase in the percentage of explants that successfully initiate and complete the regeneration process during soybean tissue culture.

3. The application according to claim 1, characterized in that, Increasing the soybean regeneration frequency refers to improving the regeneration efficiency of soybean cotyledon nodes.

4. The application according to claim 1, characterized in that, By promoting soybeans GmHRF-1 Gene expression can be used to increase soybean regeneration frequency and / or improve soybean genetic transformation efficiency.

5. The application according to claim 4, characterized in that, The promotion of soybeans GmHRF-1 The substance used for gene expression is any one of the following: C1) contains GmHRF-1 Gene expression cassettes; C2) contains GmHRF-1 Recombinant vectors of genes, or recombinant vectors containing the expression cassette described in C1); C3) contains GmHRF-1 Recombinant microorganisms containing genes, or recombinant microorganisms containing the expression cassette described in C1), or recombinant microorganisms containing the recombinant vector described in C2); C4) contains GmHRF-1 Transgenic plant cell lines containing the gene, or transgenic plant cell lines containing the expression cassette described in C1).

6. A method for improving soybean regeneration frequency and / or genetic transformation efficiency, characterized in that, Includes the following steps: soybeans GmHRF-1 Genes are linked into expression vectors to construct recombinant expression vectors. These recombinant expression vectors are then transformed into Agrobacterium competent cells to obtain Agrobacterium strains for transformation. The Agrobacterium strains are then used to infect soybean cotyledons to obtain transgenic soybean plants with improved regeneration efficiency.

7. The method according to claim 6, characterized in that, The expression vector is pPTN1171 vector, and the Agrobacterium competent cells are Agrobacterium EHA105.

8. The soybean according to claim 1 GmHRF-1 The application of the gene-encoded protein in the following (1) or (2): (1) Increase the frequency of soybean regeneration; (2) Improve the efficiency of soybean genetic transformation; The soybean GmHRF-1 The protein encoded by the gene is the protein shown in either (A1) or (A2) below: (A1) A protein consisting of the amino acid sequence shown in SEQ ID NO.2 of the sequence listing; (A2) A fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of the protein defined in (A1).

9. The application according to claim 8, characterized in that, By improving soybeans GmHRF-1 The activity of gene-encoded proteins can be used to increase soybean regeneration frequency and / or soybean genetic transformation efficiency.

10. Containing the soybean of claim 1 GmHRF-1 The application of recombinant gene expression vectors or genetically engineered bacteria in the following (1) or (2): (1) Increase the frequency of soybean regeneration; (2) Improve the efficiency of soybean genetic transformation.

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