Application of soybean GmABI4 gene in plant breeding

Abstract

The application of soybean GmABI4 gene in plant breeding, the present application relates to the field of plant genetic engineering, specifically relates to a kind of application of soybean GmABI4 gene in plant breeding.The present application provides the application of soybean GmABI4 gene in plant breeding, the nucleotide sequence of the soybean GmABI4 gene is as shown in SEQ ID NO.1 in the sequence table.The present application uses molecular means, clarifies the expression characteristics of soybean GmABI4 in the embryo of soybean development and cell nucleus specific expression, after overexpression in soybean, it is found that it can significantly improve soybean mature seed protein content, while no significant effect on grain weight.It shows that GmABI4 gene can regulate the protein content of seed.GmABI4 gene can be used to maintain soybean yield while improving soybean quality.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering, specifically to the application of the soybean GmABI4 gene in plant breeding. Background Technology

[0002] Soybean [Glycine max (L.) Merr.] is an important source of plant protein, providing up to 71% of the world's dietary protein and playing an irreplaceable role in our daily lives. Soy protein is rich in amino acids, containing all the essential amino acids required by humans, making it a high-quality complete protein with nutritional value comparable to animal protein. High-protein soybeans are a top choice for plant protein. Soy protein offers numerous benefits, including helping to lower LDL cholesterol and reducing the risk of heart disease. With rising living standards, people are increasingly focusing on healthy protein intake, and soybean protein has become a major source of healthy edible protein, with demand increasing year by year. Soybean seed protein content accounts for approximately 40% of the grain weight and is an important quality trait; increasing soybean seed protein content is one of the key breeding goals. However, in breeding practice, soybean protein and yield show a significant negative correlation. For many years, the protein content of soybean varieties targeted at yield has been decreasing, posing a significant challenge to breeding high-yield, high-quality (high-protein) soybeans. Currently, very few genes are known to regulate soybean protein, limiting the application of molecular breeding in the cultivation of high-yield, high-quality soybeans. Discovering genes that can significantly increase protein content without affecting yield is a key research objective in breeding high-yield and high-quality soybeans. Therefore, identifying genes controlling high protein content in soybeans and elucidating their molecular regulatory mechanisms are of great significance for cultivating high-yield and high-quality soybeans.

[0003] ABI4 ​​(Abscisic acid ABA-insensitive 4) belongs to the APETALA2 / ethylene response factor (AP2 / ERF) family. As a transcription factor, ABI4 has been confirmed as a positive regulator of the abscisic acid signaling pathway. The functions of ABI4 have been primarily reported in the model plant Arabidopsis thaliana, where it has been found to play important roles in several key developmental stages, including regulating seed dormancy and germination, seedling formation after seed germination, taproot and lateral root development and growth, reproductive growth transition (Chandrasekaran et al., 2020, Plant Communications), regulating salt tolerance (Kakan et al., 2021, BMC Plant Biology), and seed vigor and lifespan (Kmble et al., 2022, Planta). To date, the expression characteristics and molecular functions of the ABI4 gene in soybean remain unclear, requiring further in-depth experimental research. Summary of the Invention

[0004] The purpose of this invention is to provide the application of the soybean GmABI4 gene in plant breeding. GmABI4 has the potential to be used in the breeding of new soybean varieties and the creation of new germplasm.

[0005] This invention provides the application of the soybean GmABI4 gene in plant breeding, and the nucleotide sequence of the soybean GmABI4 gene is shown in SEQ ID NO.1 in the sequence listing.

[0006] Furthermore, the application refers to the preparation of overexpressed transgenic plants using the soybean GmABI4 gene, wherein the soybean GmABI4 gene positively regulates the protein content of plant seeds, and the plant is soybean.

[0007] Furthermore, the method for preparing the transgenic plant includes the following steps:

[0008] I. The soybean GmABI4 gene was cloned using primers to obtain the GmABI4 gene CDS sequence;

[0009] 2. The CDS sequence of the GmABI4 gene obtained in step 1 was ligated to the vector using homologous recombination to obtain a plant overexpression vector;

[0010] 3. The plant overexpression vector obtained in step 2 is introduced into the recipient plant, which is soybean, to obtain transgenic plants overexpressing the soybean GmABI4 gene.

[0011] Furthermore, in step one, the upstream primer is 5'-CCTTCACCAGAGCCTAAT-3' and the downstream primer is 5'-ATACTAAGTTCAAACCAATCG-3'.

[0012] Furthermore, the carrier described in step two is pTF101.

[0013] Furthermore, the import method described in step three includes importation using Ti plasmid, importation using Ri plasmid, importation using plant virus vector, direct DNA transformation, microinjection, or electroporation.

[0014] The present invention also provides an application of an expression vector in regulating plant protein content, wherein the expression vector contains the soybean GmABI4 gene with the nucleotide sequence shown in SEQ ID NO.1 of the sequence listing, and the plant is soybean.

[0015] The present invention also provides the application of recombinant bacteria in regulating plant protein content, wherein the recombinant bacteria contains the above-mentioned expression vector, and the plant is soybean.

[0016] The beneficial effects of this invention are:

[0017] This invention utilizes molecular methods to elucidate the expression characteristics of soybean GmABI4 in the embryo and nucleus of soybean during development. Overexpression in soybean significantly increased the protein content of mature seeds without significantly affecting seed weight. This indicates that the GmABI4 gene can regulate seed protein content. The GmABI4 gene could be used to improve soybean quality while maintaining yield.

[0018] The discovery of this gene function provides a new approach to studying the regulatory mechanism of soybean proteins, and can also accelerate the molecular breeding process of high-yield and high-quality new soybean varieties. Attached Figure Description

[0019] Figure 1 The results of PCR amplification of GmABI4 in agarose gel;

[0020] Figure 2 This analysis focuses on the relative expression levels of GmABI4 in different tissues; where Root represents root tissue; Stem represents stem tissue; Leaf represents leaf tissue; Flower represents flower tissue; SC1 represents seed coat 1 in early seed development; COT1 represents embryo 1 in early seed development; SC2 represents seed coat 2 in mid-seed development; COT2 represents embryo 2 in mid-seed development; SC3 represents seed coat 3 in late seed development; and COT3 represents embryo 3 in late seed development.

[0021] Figure 3 Subcellular localization results for GmABI4;

[0022] Figure 4 The results identified the transcriptional activation of GmABI4.

[0023] Figure 5 This is a schematic diagram of the GmABI4 overexpression vector; the GmABI4 gene is represented by a yellow rectangle, and its 5' end is connected to the CaMV 35 promoter.

[0024] Figure 6 The results are from bar test strips and PCR detection of GmABI4 transgenic overexpressing soybeans; where CK is the untransformed soybean receptor.

[0025] Figure 7 Results of GmABI4 transcription level detection in transgenic soybean lines;

[0026] Figure 8 Results of the comparison of protein content in GmABI4 transgenic soybean seeds;

[0027] Figure 9 The results of the comparison of oil content in GmABI4 transgenic soybean seeds;

[0028] Figure 10 The results show the comparison of cysteine ​​content in GmABI4-overexpressing transgenic soybean seeds and control seeds;

[0029] Figure 11 The results show the comparison of isoleucine content in GmABI4-overexpressing transgenic soybeans and control seeds;

[0030] Figure 12 The results show the comparison of lysine content in GmABI4-overexpressing transgenic soybean seeds and control seeds;

[0031] Figure 13 The results show the comparison of leucine content in GmABI4-overexpressing transgenic soybean seeds and control seeds;

[0032] Figure 14 The results show the comparison of threonine content in GmABI4-overexpressing transgenic soybean seeds and control seeds;

[0033] Figure 15 The results show the comparison of valine content in GmABI4-overexpressing transgenic soybean seeds and control seeds;

[0034] Figure 16 The results show the comparison of histidine content in GmABI4 overexpressing transgenic soybean seeds and control seeds;

[0035] Figure 17 The results show the comparison of glutamic acid content in GmABI4-overexpressing transgenic soybean seeds and control seeds;

[0036] Figure 18 The results show the comparison of glycine content in GmABI4-overexpressing transgenic soybean seeds and control seeds;

[0037] Figure 19 The results show the comparison of aspartic acid content in GmABI4 overexpressing transgenic soybean seeds and control seeds;

[0038] Figure 20 The results show the comparison of arginine content in GmABI4-overexpressing transgenic soybean seeds and control seeds;

[0039] Figure 21 The results show the comparison of tyrosine content in GmABI4-overexpressing transgenic soybean seeds and control seeds;

[0040] Figure 22 The results show the comparison of proline content in GmABI4-overexpressing transgenic soybean seeds and control seeds;

[0041] Figure 23The results show the comparison of serine content in GmABI4-overexpressing transgenic soybean seeds and control seeds;

[0042] Figure 24 The results show the comparison of 100-seed weight between GmABI4-overexpressing transgenic soybeans and the control. Detailed Implementation

[0043] The embodiments of the present invention will be described in detail below. The following embodiments are implemented based on the technical solution of the present invention, and detailed implementation schemes and specific operation processes are given. However, the protection scope of the present invention is not limited to the following embodiments.

[0044] Example 1: Cloning of the soybean GmABI4 gene

[0045] The gene sequence information of GmABI4 was retrieved from the Phytozome database. Primers for amplifying GmABI4 were designed based on the gene sequence: 5'-CCTTCACCAGAGCCTAAT-3' and 5'-ATACTAAGTTCAAACCAATCG-3'. Seeds of the soybean variety "Williams 82" (W82) were collected, ground with liquid nitrogen, and the powder was placed in a 1.5 mL EP tube. 1 mL of lysis buffer was added, and the mixture was vortexed until homogeneous. RNA extraction was then performed according to the kit (TotalRNAKit, Tiangen, China). RNA integrity was assessed by 1% agarose gel electrophoresis. Using the obtained total RNA as a template, cDNA synthesis was performed according to the instructions of the Vazyme HiScript 1st Strand cDNA Synthesis Kit (Nanjing, China). PCR amplification was then performed using the cDNA as a template. The PCR reaction mixture was as follows: 2 μL template, 2 μL each of upstream and downstream primers, 25 μL 2×PhantaMax Master Mix, and finally, ddH2O was added to bring the volume to 50 μL. The PCR program was as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 sec, 58℃ annealing for 15 sec, 72℃ extension for 60 sec, for a total of 35 cycles; final extension at 72℃ for 5 min, followed by isothermal treatment at 4℃ for 30 min. The electrophoresis results of the GmABI4 PCR amplification products are shown below. Figure 1 As shown in the figure. After gel recovery, product purification, and T-vector ligation, the product was transformed into E. coli and the target plasmid was enriched. Sequencing yielded the complete coding sequence (CDS) of the soybean GmABI4 gene, with a CDS sequence length of 1017 bp. The CDS sequence is shown in SEQ ID NO.1 in the sequence listing; the amino acid sequence of soybean GmABI4 is shown in SEQ ID NO.2.

[0046] Example 2: Tissue Expression Analysis of GmABI4 Gene

[0047] Soybean variety W82 seeds were sown in nutrient soil (peat moss: vermiculite, 2:1) and cultured in a greenhouse at 25℃ for 16h / 8h (light / dark). Seed coats (SC) and cotyledons (COT) of W82 seeds were collected from the roots (V1 stage), stems (V1 stage), leaves (V1 stage), flowers, and seeds at three key stages of nutrient accumulation (early, middle, and late stages). The samples were flash-frozen with liquid nitrogen and stored in an ultra-low temperature freezer at -80℃ for later use.

[0048] Total RNA was extracted using a plant total RNA extraction kit, and RNA integrity was assessed by 1% agarose gel electrophoresis. cDNA synthesis was performed according to the instructions of the reverse transcription kit (Vazyme). The expression level of GmABI4 in plant tissues was detected using quantitative real-time PCR (qRT-PCR). The primer sequences were 5'-TTCCACTCAAACCCTAAGACCC-3' and 5'-CCAGAAGGGCGAGGAAGC-3'. Results are shown below. Figure 2 As shown, the results indicate that GmABI4 is expressed at low levels in roots, stems, leaves, and developing seed coats, but at high levels in developing embryos (COT1, COT2, and COT3) at three stages of flower and seed development, with the highest expression level in mid-stage developing embryos (COT2), indicating that GmABI4 has a high expression characteristic in developing embryos.

[0049] Example 3: Subcellular localization of the GmABI4 gene

[0050] Specific primers were designed based on the known sequence of the GmABI4 gene and the cloning site of the plant expression vector pBSK. The primer sequences were 5'-GATAAGCTTGATATCGAATTCATGGCCTCTCTCCTTCCTCAA-3' and 5'-CATTCTAGAACTAGTGGATCCAAGATCAAAGAAGAAAGGATCATT-3'. The PCR reaction system was as follows: 2 μL template, 2 μL each of upstream and downstream primers, 25 μL 2×PhantaMax Master Mix, and finally, ddH2O was added to a final volume of 50 μL. The PCR program was as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 sec, 58℃ annealing for 15 sec, 72℃ extension for 60 sec, for a total of 35 cycles; final extension at 72℃ for 5 min, followed by isothermal incubation at 4℃ for 30 min. The target gene fragment with enzyme digestion adapters amplified by PCR was identified by gel electrophoresis, and the product was recovered from the gel. Recombinant vectors were constructed using homologous recombination. The recombination reaction was performed according to the instructions of the Vazyme ClonExpress II One Step Cloning Kit (C112). The reaction system was as follows: 1 μL vector, 2 μL target fragment, 4 μL 5×CE II Buffer, 2 μL Exnase II, and ddH2O was added to a final volume of 20 μL. The reaction program was 37℃ for 30 min. The recombinant product was transformed into DH5α competent cells using a freeze-thaw method, along with the empty vector. Colonies were plated, single clones were picked, and the cells were enriched by shaking. Subsequently, the colonies were verified by PCR sequencing. Plasmids extracted from correctly sequenced colonies were named pBSK-GmABI4. The target plasmid was transiently transformed into pre-prepared Arabidopsis protoplasts using the PEG method. The transformed protoplasts were cultured in the dark at 28℃ for 16 h, and the fluorescence excitation signal intensity of the protoplasts was observed using the Leica THUNDER system (Leica Microsystems, Wetzlar, Germany). The results are shown below. Figure 3 The first row shows the results after transformation with the empty vector plasmid, from left to right: bright field, chloroplast fluorescence channel (Chlorophyll), green fluorescence channel (GFP), and a merged image of the three channels. The second row shows the images under a microscope after transformation with the pBSK-GmABI4 vector, with the same distribution from left to right as the first row. The results indicate that the empty vector plasmid carrying GFP showed green fluorescence signals in all tissues, while the protein fluorescence signal carrying the pBSK-GmABI4 vector was mainly localized in the cell nucleus.

[0051] Example 4: Identification of GmABI4 transcriptional activity

[0052] The full-length coding region of the GmABI4 gene was ligated into the yeast expression vector pGBKT7, and then transformed into yeast AH109 competent cells. Using the empty vector pGBKT7 as a negative control, single plaques were picked and cultured in SD / -Trp liquid medium until OD2000. 600 Values ​​ranged from 0.5 to 0.6. 5 μL of each culture was incubated for 3 days on SD / -Trp and SD / -Ade-His-Trp plates. The transcriptional activity of the GmABI4 gene was detected using yeast one-hybrid technology. Figure 4 As shown, all transformed yeast cells grew normally on SD / -Trp plates, while transformed yeast cells containing GmABI4 grew normally on SD / -Trp / -His / -Ade plates, but yeast cells transformed with the empty vector pGBKT7 did not grow normally, indicating that GmABI4 has transcriptional activation activity.

[0053] Example 5: Genetic Engineering Application of the Soybean GmABI4 Gene

[0054] (I) Construction of plant overexpression vectors

[0055] Specific primers were designed based on the CDS sequence of the cloned GmABI4 gene and the cloning site of the plant expression vector pTF101. The primer sequences were 5'-GACTCTAGAAACAGAGGATCCATGGCCTCTCTCCTTCCTCAA-3' and 5'-TTCGAGCTCGCTGTTACTAGTAAGATCAAAGAAGAAAGGATCATT-3'. An overexpression vector was constructed using homologous recombination. PCR amplification was performed in a 50 μL system, containing 2 μL template DNA, 2 μL each of forward and reverse primers (10 μM), 25 μL of 2×PhantaMax Master Mix, and replenished with sterile ddH2O. The PCR program was set as follows: 95℃ pre-denaturation for 3 min; followed by 35 cycles of amplification (95℃ denaturation for 15 sec, 58-60℃ gradient annealing for 15 sec, 72℃ extension for 60 sec); and finally, a complete extension at 72℃ for 5 min, followed by storage at 4℃ for 30 min. After identification by 1% agarose gel electrophoresis, the amplified product was excised and purified by gel extraction. Recombinant plasmids were constructed using the Vazyme C112 kit. A 20 μL reaction mixture was prepared by adding 1 μL of linearized vector (50-100 ng), 2 μL of purified product (3-5 times the molar volume of the vector), 4 μL of 5×CE II Buffer, and 2 μL of Exnase II. ddH2O was added to the final volume, and the mixture was incubated at 37°C for 30 min to complete the recombination. The recombinant plasmids and empty vector controls were introduced into DH5α competent cells via freeze-thaw transformation. The cells were plated on LB agar plates containing antibiotics and cultured at 37°C for 12-16 h. Single clones were picked, and positive clones were screened by bacterial PCR and sequenced for verification. The sequencing primer sequences were 5'-CATTTCATTTGGAGAGAACACG-3' and 5'-AGCGGATAACAATTTCACACAG-3'. All critical experimental procedures were performed in a clean bench, and enzyme reaction mixtures were prepared on ice throughout the process to ensure activity. Finally, the pTF101-GmABI4 plant overexpression plasmid was obtained and stored at -20℃ for later use. A schematic diagram of the GmABI4 overexpression vector is shown below. Figure 5 The CDS sequence of the target gene GmABI4 is located between restriction endonucleases BamH1 and Spe1.

[0056] (II) Transformation of Agrobacterium tumefaciens using overexpression vector

[0057] The pTF101-GmABI4 plant overexpression plasmid obtained above was transformed into Agrobacterium tumefaciens EHA105 using a freeze-thaw method. The specific experimental procedure is as follows:

[0058] ① Use a pipette to aspirate 3 μL of the recombinant plasmid and transfer it into 100 μL of EHA105 competent cells, then gently mix.

[0059] ② Place the mixture on ice for 5 minutes, in liquid nitrogen for 5 minutes, incubate in a 37°C water bath for 5 minutes, and then immediately transfer it to an ice bath for 3 minutes.

[0060] ③ Take 800 μL of YEP culture medium with a pipette in a clean bench and revive it for 4 hours at 28℃ and 150 rpm in a constant temperature shaker.

[0061] ④ Centrifuge at 5000×g for 2 min at room temperature, resuspend the bacterial cells with an appropriate amount of supernatant, take 200μL of the revived bacterial solution and spread it evenly on the screening plate medium, and incubate at 28℃ upside down for 3 days.

[0062] (III) Transformation of soybean cotyledon nodes

[0063] High-quality W82 soybean seeds were selected and sterilized in a fume hood using chlorine sterilization for 16 hours. The sterile seeds were then soaked in sterile water and cultured in a tissue culture room at 25°C for 24 hours. After imbibition, the germinating soybeans were longitudinally split along the midline, and the apical and axillary buds were removed. Slight incisions were made at the cotyledonary nodes. The transformed Agrobacterium-mediated transformation solution was shaken to OD... 600 Centrifuge at 0.5 μL, 12000 rpm, resuspend in the infusion solution, and place the prepared cotyledonary explants in it, allowing them to stand at 28°C for 30 min. Dry the infused cotyledonary explants by aspirating the bacterial solution, place them adaxially on CCM solid medium, arrange them neatly, and incubate in the dark in a tissue culture room for 3 days. Wash the cotyledonary nodes three times in sterile water, blot off the liquid with sterile filter paper, and insert them onto recovery solid medium, incubating in a tissue culture room for 15 days. Cut off resistant shoots that have grown to 1-2 cm and insert them into elongation medium, incubating in a tissue culture room for 15 days. Once the elongated seedlings are robust, insert them into rooting medium and culture upright for 10 days. When the elongated seedlings have developed sufficient roots, transplant them into humus mixed with vermiculite, and then transfer them to a greenhouse for further cultivation.

[0064] (iv) Identification of positive transformation and gene expression analysis

[0065] The overexpression tissue culture seedlings transplanted into soil were first subjected to bar gene detection (PAT / bar test strips), and the results are shown below. Figure 6As shown in Figure a, the untransformed soybean plants (CK) only showed one control band, with no detection band, while all three transgenic overexpression lines showed detection bands (red arrows). DNA was extracted from leaves of the overexpression tissue culture seedlings after the initial detection. PCR identification was performed using Novizan polymerase (2×RapidTaq Master Mix). The PCR system was as follows: 10 μL of 2×RapidTaq Master Mix, 2 μL of Primer 1 (10 μM), 2 μL of Primer 2 (10 μM), 2 μL of Template DNA, and finally, ddH2O was added to a final volume of 20 μL. The reaction program was as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 sec, 60℃ annealing for 15 sec, 72℃ extension for 15 sec, for a total of 35 cycles; 72℃ final extension for 5 min. Seedlings with PCR bands matching the specified size were considered positive for overexpression. Results are shown in [Figure number missing]. Figure 6 b. Next, qRT-PCR was used to further examine the GmABI4-OE transgenic soybean plants. The results showed that the expression level of GmABI4 in the GmABI4-OE overexpressing transgenic soybean plants was significantly higher than that in the control W82 (**P<0.01). Figure 7 Bar test strips, PCR molecular detection, and qRT-PCR experiments all confirmed that the recombinant plasmid was successfully transferred into soybeans and expressed normally.

[0066] (V) Determination of Protein Content in Genetically Modified Soybeans

[0067] The harvested transgenic soybean seeds were dried in a 30°C oven. Then, the protein and oil content of mature seeds from the control material W82 and GmABI4-OE transgenic soybean plants were determined using an Antaris II Fourier transform near-infrared spectrometer (Thermo Fisher Scientific, USA). Three independent transformant lines were selected, and 5-8 individual plants from each line were measured. The comparison results of protein and oil content in GmABI4 overexpression transgenic soybean seeds and control seeds are shown below. Figure 8 and Figure 9 As shown in the figure (where the error bars represent the mean ± SD, and the statistical analysis was performed using a two-tailed t-test, **, P < 0.01; ***, P < 0.001), the results showed that, compared with the control group, the three lines that overexpressed GmABI4 significantly increased the protein content by 7.68%-8.06%, while the oil content significantly decreased by 4.24%-5.55%.

[0068] (vi) Determination of amino acid content in genetically modified soybean seeds

[0069] The amino acid content in dried seeds of transgenic soybean generation T3 was determined using a Porton DA7250 near-infrared spectrometer (Perten, Sweden). Three independent transgenic lines were used in the experiment, and 5-8 individual plants from each line were analyzed. The comparison results of amino acid content in GmABI4 overexpressing transgenic soybean seeds and control seeds are shown below. Figures 10-23 As shown in the figure (where the error bars represent the mean ± SD, and the statistical analysis was performed using a two-tailed t-test, **, P < 0.01; ***, P < 0.001), the results showed that the contents of 14 amino acids (cysteine, lysine, leucine, threonine, valine, isoleucine, glycine, glutamic acid, arginine, tyrosine, proline, serine, aspartic acid, and histidine) in the three lines overexpressing GmABI4 were significantly increased compared with those in the unconverted soybean W82. Among them, the content of sulfur-containing amino acids (cysteine) increased by 9.5%, and the content of the five essential amino acids isoleucine, lysine, leucine, threonine and valine increased by 4.68%-6.02%, 4.31%-5.86%, 5.27%-6.71%, 4.17%-6.40% and 4.88%-6.07%, respectively. Histidine, glutamic acid, glycine, aspartic acid, arginine, tyrosine, proline and serine increased by 5.33%-5.86%, 6.87%-9.45%, 5.87%-7.42%, 6.08%-8.26%, 8.35%-10.71%, 4.17%-5.97%, 5.47%-7.20% and 6.56%-9.49%, respectively.

[0070] (vii) Determination of the weight of 100 genetically modified soybeans

[0071] The harvested transgenic soybean seeds were dried in an oven at 30℃. Then, the 100-seed weight of mature seeds from the control materials W82 and GmABI4-OE transgenic soybean plants was measured. Three independent transformant lines were used in the experiment, and 5-8 individual plants from each transformant line were measured. The comparison of the 100-seed weight of GmABI4 overexpressing transgenic soybeans with the control is shown below. Figure 24 As shown in the figure (where the error bars represent the mean ± SD, and the statistical analysis was performed using a two-tailed t-test, **, P < 0.01; ***, P < 0.001), the results showed that, compared with the control, the 100-grain weight of the three lines overexpressing GmABI4 was significantly reduced by 11.02%-14.97%.

Claims

1. Application of overexpression of soybean GmABI4 gene in soybean breeding to improve soybean seed protein content, wherein the nucleotide sequence of soybean GmABI4 gene is shown in SEQ ID NO.

1.

2. The application according to claim 1, characterized in that, Includes the following steps: I. The soybean GmABI4 gene was cloned using primers to obtain the GmABI4 gene CDS sequence; 2. The CDS sequence of the GmABI4 gene obtained in step 1 was ligated to the vector using homologous recombination to obtain a plant overexpression vector; 3. The plant overexpression vector obtained in step 2 is introduced into a recipient plant, namely soybean, to obtain a transgenic plant overexpressing the soybean GmABI4 gene.

3. The application according to claim 2, characterized in that, In step one, the upstream primer is 5'-CCTTCACCAGAGCCTAAT-3' and the downstream primer is 5'-ATACTAAGTTCAAACCAATCG-3'.

4. The application according to claim 2, characterized in that, The carrier mentioned in step two is pTF101.

5. The application according to claim 2, characterized in that, The import methods described in step three include importation using Ti plasmids, importation using Ri plasmids, importation using plant virus vectors, direct DNA transformation, microinjection, or electroporation.

6. The application of an expression vector in increasing the protein content of soybean seeds, wherein the expression vector contains the soybean GmABI4 gene with the nucleotide sequence shown in SEQ ID NO.

1.

7. The application of a recombinant bacterium in increasing the protein content of soybean seeds, said recombinant bacterium containing the expression vector of claim 6.

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