Application of soybean GmABI4 gene in plant breeding

Abstract

The invention discloses application of a soybean GmABI4 gene in plant breeding, and relates to the field of plant genetic engineering, in particular to application of the soybean GmABI4 gene in plant breeding. The invention provides an application of a soybean GmABI4 gene in plant breeding. A nucleotide sequence of the soybean GmABI4 gene is shown as SEQ ID NO.1 in a sequence table. According to the invention, the expression characteristics of specific expression and cell nucleus specific expression of soybean GmABI4 in soybean developed embryos are illustrated by using a molecular means, and after over-expression in soybeans, it is found that the soybean GmABI4 can significantly increase the protein content of soybean mature seeds and has no significant influence on the grain weight. The result shows that the GmABI4 gene can regulate and control the protein content of the seeds. The GmABI4 gene can be used for improving the soybean quality while maintaining the soybean yield.

Description

Technical Field

[0001] The present invention relates to the field of plant genetic engineering, and particularly relates to the application of a soybean GmABI4 gene in plant breeding. Background Art

[0002] Soybean [Glycine max (L.) Merr.] is one of the important sources of plant protein, providing up to about 71% of the protein for the world's diet and playing an irreplaceable role in our daily life. The amino acid composition of soybean protein is rich, containing the amino acids required by humans, belonging to high-quality complete protein, and its nutritional value can be equivalent to that of animal protein. High-content soybean protein is the first choice of plant protein. Soybean protein has many advantages such as helping to reduce low-density lipoprotein cholesterol and reducing the risk of heart disease. With the increasing living standards of people, the daily intake of healthy protein has gradually attracted people's attention. Soybean protein has become the main source of healthy edible protein, and the demand is increasing year by year. The protein content in soybean seeds accounts for about 40% of the grain weight, which is an important quality trait of soybeans. Increasing the protein content in soybean seeds is one of the important breeding goals. However, in breeding practice, there is a significant negative correlation between soybean protein and yield. The protein content in soybean varieties targeted for yield has been decreasing year by year, which brings great difficulties to the cultivation of high-yield and high-quality (high-protein) soybeans. At present, very few genes regulating soybean protein are known, which limits the application of molecular breeding in the cultivation of high-yield and high-quality soybeans. Discovering control genes that can significantly increase the protein content without affecting the yield is an important research goal in the cultivation of high-yield and high-quality soybeans at present. Therefore, identifying soybean high-protein control genes and clarifying the molecular regulation mechanism are of great significance for the cultivation of 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 to be a positive regulator of the abscisic acid signaling pathway. The functions of ABI4 have been mainly reported in the model plant Arabidopsis thaliana, and it is found that it plays important roles in multiple key developmental stages of plants, mainly including regulating seed dormancy and germination, seedling establishment after seed germination, the occurrence and development of main roots and lateral roots, reproductive growth transition (Chandrasekaran et al, 2020, Plant Communications), regulating salt tolerance (Kakan et al, 2021, BMC Plant Biology), seed vigor and lifespan (Kmble et al, 2022, Planta). So far, the expression characteristics and molecular functions of the ABI4 gene in soybeans are still unclear and in urgent need of in-depth experimental research. Summary of the Invention

[0004] The object of the present invention is to provide the application of soybean GmABI4 gene in plant breeding, and GmABI4 has the potential for the breeding of new soybean varieties and the creation of new germplasms.

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

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

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

[0008] I. Cloning the soybean GmABI4 gene using primers to obtain the CDS sequence of the GmABI4 gene;

[0009] II. Connecting the CDS sequence of the GmABI4 gene obtained in step I with a vector using homologous recombination to obtain a plant overexpression vector;

[0010] III. Introducing the plant overexpression vector obtained in step II into a receptor plant, which is soybean, to obtain a transgenic plant overexpressing the soybean GmABI4 gene.

[0011] Furthermore, the upstream primer in the primers in step I is 5’-CCTTCACCAGAGCCTAAT-3’, and the downstream primer is 5’-ATACTAAGTTCAAACCAATCG-3’.

[0012] Furthermore, the vector in step II is pTF101.

[0013] Furthermore, the introduction method in step III is Ti plasmid introduction, Ri plasmid introduction, plant virus vector introduction, direct DNA transformation, microinjection or electroporation.

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

[0015] The present invention also provides the application of a recombinant bacterium in regulating the protein content in plants. The recombinant bacterium contains the above expression vector, and the plant is soybean.

[0016] The beneficial effects of the present invention:

[0017] The present invention uses molecular means to clarify the expression characteristics of soybean GmABI4, which is specifically expressed in developing embryos of soybeans and specifically expressed in the cell nucleus. After overexpression in soybeans, it was found that it can significantly increase the protein content of mature soybean seeds, while having no significant effect on seed weight. This indicates that the GmABI4 gene can regulate the protein content of seeds. The GmABI4 gene can be used to improve the quality of soybeans while maintaining soybean yield.

[0018] The discovery of this gene function provides a new way to study the mechanism of soybean protein regulation, and at the same time can accelerate the molecular breeding process of new high-yield and high-quality soybean varieties. Brief Description of the Drawings

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

[0020] Figure 2 Analysis of the relative tissue expression level of GmABI4; where Root: root tissue; Stem: stem tissue; Leaf: leaf tissue; Flower: flower tissue; SC1: seed coat (Seed Coat1) tissue in the early stage of developing seeds; COT1: embryo (Cotyledon1) tissue in the early stage of developing seeds; SC2: seed coat (Seed Coat2) tissue in the middle stage of developing seeds; COT2: embryo (Cotyledon2) tissue in the middle stage of developing seeds; SC3: seed coat (Seed Coat3) tissue in the late stage of developing seeds; COT3: embryo (Cotyledon3) tissue in the late stage of developing seeds;

[0021] Figure 3 Results of subcellular localization of GmABI4;

[0022] Figure 4 Results of transcriptional activation identification of GmABI4;

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

[0024] Figure 6 Results of bar strip test and PCR test of GmABI4 transgenic overexpressing soybeans; where CK is the untransformed soybean receptor;

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

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

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

[0028] Figure 10 The comparison result of cysteine content between GmABI4 overexpressing transgenic soybean and control seeds;

[0029] Figure 11 The comparison result of isoleucine content between GmABI4 overexpressing transgenic soybean and control seeds;

[0030] Figure 12 The comparison result of lysine content between GmABI4 overexpressing transgenic soybean and control seeds;

[0031] Figure 13 The comparison result of leucine content between GmABI4 overexpressing transgenic soybean and control seeds;

[0032] Figure 14 The comparison result of threonine content between GmABI4 overexpressing transgenic soybean and control seeds;

[0033] Figure 15 The comparison result of valine content between GmABI4 overexpressing transgenic soybean and control seeds;

[0034] Figure 16 The comparison result of histidine content between GmABI4 overexpressing transgenic soybean and control seeds;

[0035] Figure 17 The comparison result of glutamic acid content between GmABI4 overexpressing transgenic soybean and control seeds;

[0036] Figure 18 The comparison result of glycine content between GmABI4 overexpressing transgenic soybean and control seeds;

[0037] Figure 19 The comparison result of aspartic acid content between GmABI4 overexpressing transgenic soybean and control seeds;

[0038] Figure 20 The comparison result of arginine content between GmABI4 overexpressing transgenic soybean and control seeds;

[0039] Figure 21 The comparison result of tyrosine content between GmABI4 overexpressing transgenic soybean and control seeds;

[0040] Figure 22 The comparison result of proline content between GmABI4 overexpressing transgenic soybean and control seeds;

[0041] Figure 23Comparison results of serine content in GmABI4 overexpressing transgenic soybeans and control seeds;

[0042] Figure 24 Comparison results of 100-seed weight between GmABI4 overexpressing transgenic soybeans and the control. Detailed implementation mode

[0043] The following will give a detailed description of the embodiments of the present invention. The following embodiments are implemented on the premise of 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 soybean GmABI4 gene

[0045] According to the gene sequence information of GmABI4 found in the Phytozome database, primers for amplifying GmABI4 were designed. The primer sequences were 5’-CCTTCACCAGAGCCTAAT-3’ and 5’-ATACTAAGTTCAAACCAATCG-3’. Taking the soybean variety "Williams 82" (W82) as the material, its developing seeds were taken, ground with liquid nitrogen, and the powder was put into a 1.5 mL EPP tube. 1 mL of lysis buffer was added, and it was vortexed to mix evenly. Then, extraction was carried out according to the kit (Total RNA Kit, Tiangen, China). The integrity of RNA was detected by 1% agarose gel electrophoresis. Using the obtained total RNA as a template, cDNA synthesis was carried out according to the instructions of the reverse transcription kit provided by Vazyme Company (Vazyme HiScript 1st Strand cDNA Synthesis Kit, Nanjing, China). PCR amplification was carried out using cDNA as a template. The PCR reaction system was as follows: 2 μL of template, 2 μL each of upstream and downstream primers, 25 μL of 2×PhantaMax Master Mix, and finally ddH2O was added to make up the volume to 50 μL. The PCR program was as follows: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 sec, annealing at 58°C for 15 sec, extension at 72°C for 60 sec, for a total of 35 cycles; finally, complete extension at 72°C for 5 min, and then incubation at 4°C for 30 min. The electrophoresis results of the PCR amplification product of GmABI4 are as Figure 1 shown. After gel recovery, product purification and T-vector ligation, it was transferred into Escherichia coli and the target plasmid was enriched. After sequencing, the sequence of the soybean GmABI4 gene with the complete coding sequence (CDS) was obtained. The CDS sequence length was 1017 bp. The CDS sequence is shown as SEQ ID NO.1 in the sequence listing; the amino acid sequence of soybean GmABI4 is shown as SEQ ID NO.2.

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

[0047] Sow the seeds of soybean variety W82 in nutrient soil (peat soil: vermiculite, 2:1), place them in a greenhouse for cultivation, and the cultivation environment is 25°C, 16h / 8h (light / dark). Respectively take the roots (V1 stage), stems (V1 stage), leaves (V1 stage), flowers, and the seed coats (SC) and cotyledons (COT) at three key stages of seed nutrient accumulation (early stage, middle stage, late stage) of W82. Quick-freeze the samples with liquid nitrogen and store them in a -80°C ultra-low temperature refrigerator for later use.

[0048] Extract total RNA using a plant total RNA extraction kit, and detect the integrity of RNA by 1% agarose gel electrophoresis. The synthesis of cDNA is carried out according to the instructions of the reverse transcription kit (Vazyme). Detect the expression level of GmABI4 in plant tissues using real-time fluorescence quantitative PCR (qRT-PCR). The primer sequences are 5’-TTCCACTCAAACCCTAAGACCC-3’ and 5’-CCAGAAGGGCGAGGAAGC-3’. The results are as Figure 2 shown. The results show that the expression level of GmABI4 is relatively low in roots, stems, leaves and developing seed coats, while it is relatively high in flowers and developing embryos (COT1, COT2, COT3) at three stages of seed development, and the expression level is the highest in the middle-stage developing embryo (COT2), indicating that GmABI4 has the characteristic of high expression in developing embryos.

[0049] Example 3: Subcellular Localization of GmABI4 Gene

[0050] Specific primers were designed according to the known sequence of the GmABI4 gene and the cloning sites 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 of template, 2 μL each of the upstream and downstream primers, 25 μL of 2×PhantaMax Master Mix, and finally ddH2O was added to make the volume up to 50 μL. The PCR program was as follows: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 sec, annealing at 58°C for 15 sec, extension at 72°C for 60 sec, for a total of 35 cycles; finally, complete extension at 72°C for 5 min, and then incubation at 4°C for 30 min. The target gene fragment with restriction enzyme adaptors amplified by PCR was identified by gel electrophoresis, and the obtained product was subjected to gel extraction. The recombinant vector was constructed by homologous recombination method. The recombination reaction was carried out according to the instructions of the ClonExpressII One Step Cloning Kit (C112) of Vazyme company. The reaction system was as follows: 1 μL of vector, 2 μL of target fragment, 4 μL of 5×CE II Buffer, 2 μL of Exnase II, and ddH2O was added to make the volume up to 20 μL. The reaction program: recombination reaction at 37°C for 30 min. The recombinant product and the empty plasmid were transformed into DH5α competent cells by the freeze-thaw method respectively. The plates were coated, single colonies were picked, and the target colonies were enriched by shaking the bacteria. Subsequently, the bacterial liquid was verified by PCR sequencing. The plasmid of the bacterial liquid with correct sequencing was extracted and named pBSK-GmABI4. The target plasmid was transiently transformed into the pre-prepared Arabidopsis protoplasts by the PEG method. The transformed protoplasts were cultured in the dark at 28°C for 16 h, and then the fluorescence excitation signal intensity of the protoplasts was observed using the Leica THUNDER system (Leica Microsystems, Wetzlar, Germany). The results are shown in Figure 3 , where the first row is the result after transformation with the empty plasmid. From left to right, they are bright field (Bright), chloroplast fluorescence channel (Chlorophyll), green fluorescence channel (GFP), and the merged image of the above three channels (Merged); the second row of the picture shows the imaging under the microscope after transformation with the pBSK-GmABI4 vector. The picture distribution from left to right is the same as the first row. The results show that the empty vector plasmid carrying GFP has green fluorescence signals in all tissues, while the protein fluorescence signals of the vector carrying pBSK-GmABI4 are mainly localized in the nucleus.

[0051] Example 4: Identification of GmABI4 transcriptional activity

[0052] The full-length fragment of the coding region of the GmABI4 gene was ligated to the yeast expression vector pGBKT7, and then transformed into competent yeast AH109 cells. Using the empty vector pGBKT7 as a negative control, single colonies were picked and cultured in SD / -Trp liquid medium until the OD 600 value reached 0.5 - 0.6. Then, 5 μL of each culture was taken and cultured on SD / -Trp and SD / -Ade-His-Trp plate media for 3 days. The transcriptional activity of the GmABI4 gene was detected using the yeast one-hybrid technique. As Figure 4 shown, on the SD / -Trp plate, all transformed yeast cells could grow normally. However, on the SD / -Trp / -His / -Ade plate, the transformed yeast cells containing GmABI4 could grow normally, while the yeast cells transformed with the empty vector pGBKT7 could 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 vector

[0055] Specific primers were designed according to the CDS sequence of the cloned GmABI4 gene and the cloning sites of the plant expression vector pTF101. The primer sequences were 5’-GACTCTAGAAACAGAGGATCCATGGCCTCTCTCCTTCCTCAA-3’ and 5’-TTCGAGCTCGCTGTTACTAGTAAGATCAAAGAAGAAAGGATCATT-3’. An overexpression vector was constructed by homologous recombination. A 50 μL system was used for PCR amplification, which included 2 μL of template DNA, 2 μL each of upstream and downstream primers (10 μM), 25 μL of 2×Phanta Max Master Mix, and the volume was made up with sterile ddH2O. The PCR program was set as follows: pre-denaturation at 95 °C for 3 min; then 35 cycles of amplification reaction (denaturation at 95 °C for 15 sec, gradient annealing at 58 - 60 °C for 15 sec, extension at 72 °C for 60 sec); finally, thorough extension at 72 °C for 5 min and preservation at 4 °C for 30 min. After the amplified product was identified by 1% agarose gel electrophoresis, the target band was cut out for gel recovery and purification. The Vazyme C112 kit was used to construct the recombinant plasmid. According to the 20 μL reaction system, 1 μL of linearized vector (50 - 100 ng), 2 μL of purified product (3 - 5 times the molar amount of the vector), 4 μL of 5×CE II Buffer, and 2 μL of Exnase II were added, and ddH2O was added to the final volume. The reaction was carried out at 37 °C for 30 min to complete the recombination. The recombinant plasmid and the empty vector control were respectively introduced into DH5α competent cells by the freeze-thaw transformation method. The LB plates containing antibiotics were coated and cultured at 37 °C for 12 - 16 h. Then single colonies were picked, and positive clones were screened by colony PCR and sent for sequencing verification. The sequencing primer sequences were 5’-CATTTCATTTGGAGAGAACACG-3’ and 5’-AGCGGATAACAATTTCACACAG-3’. All key experimental operations were completed in a laminar flow hood, and the enzyme reaction system was prepared on ice throughout the process to ensure activity. Finally, the pTF101-GmABI4 plant overexpression plasmid was obtained and stored at -20 °C in the refrigerator for later use. The schematic diagram of the GmABI4 overexpression vector is shown in Figure 5 The CDS sequence of the target gene GmABI4 is between the restriction enzymes BamH1 and Spe1.

[0056] (2) Transformation of the overexpression vector into Agrobacterium

[0057] The pTF101-GmABI4 plant overexpression plasmid obtained above was transformed into Agrobacterium tumefaciens EHA105 by the freeze-thaw method. The specific experimental operation process 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, and gently mix.

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

[0060] ③ Using a pipette, take 800 μL of YEP medium in a laminar flow hood and resuscitate it at 28 °C and 150 rpm in a constant temperature shaker for 4 h.

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

[0062] (III) Transformation of soybean cotyledon nodes

[0063] Select high-quality W82 soybean seeds and sterilize them using chlorine gas sterilization method in a fume hood for 16 h. Soak the sterile seeds in sterile water and culture them at 25 °C in a tissue culture room for 24 h. Longitudinally cut the imbibed germinated soybeans along the midline, remove the apical buds and axillary buds, and make slight scratches at the cotyledon nodes. Shake the transformed Agrobacterium liquid until OD 600 = 0.5, centrifuge at 12000 rpm, resuspend with the infection liquid, and place the scratched cotyledon node explants into it, and let it stand at 28 °C for 30 min. Blot the infected cotyledon node explants to remove the bacterial liquid, place the adaxial surface on the CCM solid medium, arrange them neatly, and culture them in the dark in a tissue culture room for 3 d. Wash the cotyledon nodes 3 times in sterile water, blot the liquid with sterilized filter paper, insert them onto the recovery solid medium, and culture them in the tissue culture room for 15 d. Cut the resistant buds that have grown to 1 - 2 cm and insert them into the elongation medium, and culture them in the tissue culture room for 15 d. When the elongated seedlings grow strong, insert them into the rooting medium and culture them upright for 10 d. When the elongated seedlings grow an appropriate amount of roots, transplant them into humus soil mixed with vermiculite, and then transfer them to the greenhouse for cultivation.

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

[0065] The overexpressed tissue culture seedlings transplanted into the soil are first tested for the bar gene (PAT / bar test strip), and the results are shown in Figure 6As shown in a of Figure 6 , the untransformed soybean plants (CK) only showed one quality control band and no detection band, while the three transgenic overexpression lines all showed detection bands (red arrows); after detection, the leaves of the overexpressed tissue culture seedlings were taken to extract DNA. PCR identification was carried out using Novoprotein polymerase (2×RapidTaq Master Mix). The PCR system was as follows: 2×Rapid Taq MasterMix 10 μL, Primer 1 (10 μM) 2 μL, Primer 2 (10 μM) 2 μL, Template DNA 2 μL, and finally made up to 20 μL with ddH2O. The reaction program was as follows: pre-denaturation at 95 °C for 3 min; denaturation at 95 °C for 15 sec, annealing at 60 °C for 15 sec, extension at 72 °C for 15 sec, for a total of 35 cycles; final extension at 72 °C for 5 min. The seedlings with PCR identification bands of the appropriate size were overexpression positive seedlings. The results are shown in Figure 7 . The bar test strip, 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 transgenic soybeans

[0067] The harvested transgenic soybean seeds were dried in an oven at 30 °C, and then the protein and oil contents of the mature seeds of the control material W82 and GmABI4-OE transgenic soybean plants were determined using an Antaris II Fourier transform near-infrared spectrometer produced by Thermo Fisher Scientific (USA). Three independent transgenic lines were selected for the experiment, and 5-8 individual plants were measured for each line. The comparison results of the protein and oil contents in GmABI4 overexpressing transgenic soybeans and control seeds are shown in Figure 8 and Figure 9 (where the error bars represent mean ± SD, and 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 protein content in the three lines overexpressing GmABI4 increased significantly by 7.68% - 8.06%, while the oil content decreased significantly by 4.24% - 5.55%.

[0068] (VI) Determination of amino acid content in transgenic soybean seeds

[0069] The amino acid content in the dry seeds of the T3 generation of transgenic soybeans was determined using a DA7250 near-infrared analyzer (Perten, Sweden). Three independent transgenic lines were used in the experiment, and 5 - 8 individual plants were measured for each transgenic line. The comparison results of the amino acid content between the GmABI4 overexpressing transgenic soybeans and the control seeds are as Figures 10 - 23 shown (where the error bars represent 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, histidine) in the three lines overexpressing GmABI4 were all significantly increased compared with the untransformed receptor soybean W82. Among them, the content of sulfur-containing amino acid (cysteine) increased by 9.5%, and the contents 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%, 4.88% - 6.07% respectively. The contents of 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%, 6.56% - 9.49% respectively.

[0070] (VII) Determination of 100-seed weight of transgenic soybeans

[0071] The harvested transgenic soybean seeds were dried in an oven at 30 °C, and then the 100-seed weight of the mature grains of the control material W82 and the GmABI4-OE transgenic soybean plants was measured. Three independent transgenic lines were used in the experiment, and 5 - 8 individual plants were measured for each transgenic line. The comparison results of the 100-seed weight between the GmABI4 overexpressing transgenic soybeans and the control are as Figure 24 shown (where the error bars represent 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 100-seed weight of the three lines overexpressing GmABI4 was significantly reduced by 11.02% - 14.97% compared with the control.

Claims

1. Use of the soybean GmABI4 gene in plant breeding, wherein the nucleotide sequence of the soybean GmABI4 gene is as shown in SEQ ID NO.1 in the sequence listing.

2. The application according to claim 1, characterized in that The use refers to preparing an overexpressing transgenic plant using the soybean GmABI4 gene. The soybean GmABI4 gene positively regulates the protein content in plant seeds, and the plant is soybean.

3. The application according to claim 2, wherein The method for preparing the transgenic plant comprises the following steps: First, clone the soybean GmABI4 gene using primer pairs to obtain the CDS sequence of the GmABI4 gene; Second, use the method of homologous recombination to ligate the CDS sequence of the GmABI4 gene obtained in the first step with a vector to obtain a plant overexpression vector; Third, introduce the plant overexpression vector obtained in the second step into a recipient plant, which is soybean, to obtain a transgenic plant overexpressing the soybean GmABI4 gene.

4. The application according to claim 3, characterized in that, The upstream primer in the primer pairs in the first step is 5’-CCTTCACCAGAGCCTAAT-3’, and the downstream primer is 5’-ATACTAAGTTCAAACCAATCG-3’.

5. The application according to claim 3, wherein The vector in the second step is pTF101.

6. The application according to claim 3, wherein The introduction method in the third step is introduction using a Ti plasmid, introduction using an Ri plasmid, introduction using a plant virus vector, direct DNA transformation, microinjection, or electroporation method.

7. Use of an expression vector in regulating the protein content in plants, wherein the expression vector contains the soybean GmABI4 gene with a nucleotide sequence as shown in SEQ ID NO.1 in the sequence listing, and the plant is soybean.

8. Use of a recombinant bacterium in regulating the protein content in plants, wherein the recombinant bacterium contains the above-mentioned expression vector, and the plant is soybean.

Images