Application of GmABI3 protein in increasing soybean protein content or decreasing soybean oil content

Abstract

The present invention discloses the application of GmABI3 protein in increasing the protein content of soybeans or decreasing the oil content of soybeans. It belongs to the technical field of plant breeding. The purpose of the present invention is to improve the quality of soybeans, including the protein content and oil content of soybeans. The present invention provides the application of GmABI3 protein in increasing the protein content of soybeans or decreasing the oil content in soybeans, providing a theoretical basis for the subsequent preparation of soybeans with high protein yield and high oil content.

Description

Technical Field

[0001] The invention belongs to the technical field of plant breeding, and particularly relates to the application of GmABI3 protein in increasing soybean protein content or reducing soybean oil content. Background Art

[0002] As global consumer demand for high-quality soy products continues to grow, improving soybean quality is becoming increasingly important. Mature soybean kernels contain approximately 40% protein and 20% oil, respectively, of their dry weight. Soy protein, with its excellent gelling properties and low cost, can be used as a plant-based meat substitute. Soybeans are rich in nutrients, and consumers are increasingly focused on eating well rather than just filling themselves up. Therefore, improving soybean varieties and enhancing soybean quality are crucial. However, since protein and oil content are quantitative traits controlled by multiple genes and influenced by environmental factors, improving varieties presents a greater challenge. Furthermore, soybean protein and oil content are negatively correlated, so finding germplasm resources that improve one while maintaining the other presents significant challenges.

[0003] my country's soybean production requires more effective methods for improvement. Because soybeans are a paleotetraploid crop with multiple copies of genes, they are limited by genetic redundancy. Consequently, forward genetic studies on soybeans have yielded limited results compared to other model plant species. my country urgently needs to increase soybean protein content and cultivate high-protein, high-oil soybean varieties to meet people's daily needs. Summary of the Invention

[0004] The purpose of the present invention is to improve the quality of soybeans, including the protein content and oil content of soybeans.

[0005] The present invention provides an application of GmABI3 protein in increasing soybean protein content or reducing soybean oil content.

[0006] It is further defined that the sequence of the GmABI3 protein is shown as SEQ ID NO.2.

[0007] The present invention provides an application of a GmABI3 gene in increasing soybean protein content or reducing soybean oil content.

[0008] It is further defined that the sequence of the GmABI3 gene is shown as SEQ ID NO.1.

[0009] The present invention provides an application of a vector containing a GmABI3 gene in increasing soybean protein content or reducing soybean oil content. The sequence of the GmABI3 gene is shown in SEQ ID NO.1.

[0010] The present invention provides an application of a recombinant microbial cell containing a GmABI3 gene in increasing soybean protein content or reducing soybean oil content. The sequence of the GmABI3 gene is shown in SEQ ID NO.1.

[0011] The present invention provides a method for increasing soybean oil content, the method comprising the following steps:

[0012] (1) Amplify the sgRNA sequence and insert the gene sequence into the vector; the sgRNA sequence is shown in SEQ ID NO.3;

[0013] (2) introducing the vector obtained in step (1) into Agrobacterium, and using Agrobacterium to transform into soybean to obtain transgenic soybean;

[0014] (3) Identifying the transgenic soybeans obtained in step (2) to obtain positive transgenic soybeans.

[0015] The present invention provides an application of a soybean plant containing a knocked-out GmABI3 gene in increasing the oil content of soybeans.

[0016] The present invention provides a soybean plant containing an over-expressed GmABI3 gene for use in increasing soybean protein content or increasing soybean plant height.

[0017] The present invention provides a method for increasing the protein content of soybeans, characterized in that the method comprises the following steps:

[0018] (1) Amplify the GmABI3 gene sequence and insert the gene sequence into the overexpression vector;

[0019] (2) introducing the vector obtained in step (1) into Agrobacterium, and using Agrobacterium to transform into soybean to obtain transgenic soybean;

[0020] (3) Identifying the transgenic soybeans obtained in step (2) to obtain positive transgenic soybeans.

[0021] Beneficial Effects: Construction of a Gmabi3 mutant and analysis of its protein and oil content revealed an average 3.96% decrease in protein content and a 2.06% increase in oil content compared to the WT. Transmission electron microscopy also revealed a trend toward a decrease in the number of protein bodies and an increase in the number of oil bodies. Grains of GmABI3-overexpressing materials showed an average 2.56% increase in protein content and a 2.36% decrease in oil content compared to the WT. Transmission electron microscopy also observed an increase in the number of protein bodies and a decrease in the number of oil bodies. This demonstrates that GmABI3 positively regulates soybean seed protein content and negatively regulates oil content. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1For the construction of pGES201-GmABI3 mutant vector, M: Trans 2K Plus DNAMaker; 1-4: PCR amplification products of E. coli transformed with pGES201-GmABI3 plasmid;

[0023] Figure 2 is the PCR amplification product of soybean GmABI3, M is Trans 2K Plus DNAMaker; 1-8 are the PCR amplification results of GmABI3 gene;

[0024] Figure 3 is the PCR amplification product of E. coli transformed with FU28-GmABI3 plasmid, M is Trans 2K Plus DNA Maker, positive control: plasmid, negative control: water, 1-8 are the PCR amplification products of E. coli transformed with FU28-GmABI3 plasmid;

[0025] Figure 4 is the PCR amplification product of Agrobacterium transformed with pSOY1-GmABI3 plasmid, M is Trans 2K Plus DNA Maker, positive control: plasmid, negative control: water, 1-7 are the PCR amplification products of Agrobacterium transformed with pSOY1-GmABI3 plasmid;

[0026] Figure 5 This is a flowchart of soybean genetic transformation;

[0027] Figure 6 Schematic diagram of Gmabi3 mutants;

[0028] Figure 7 Figure 1 shows the results of PCR testing of overexpressing plants. M: Trans 2K Plus DNA Marker; 1: GmABI3 plasmid; 2: Dongnong 50; 3-13: transgenic plants;

[0029] Figure 8 Figures 1-6: transgenic plants;

[0030] Figure 9 Western blot detection of transgenic plants;

[0031] Figure 10 are the total nitrogen content, total fatty acid content, and fatty acid content of the transgenic plants;

[0032] Figure 11 This is the result of transmission electron microscopy observation of transgenic soybean grains;

[0033] Figure 12 These are the agronomic traits of T4 generation transgenic soybean plants. DETAILED DESCRIPTION

[0034] Example 1. Construction of GmABI3 mutant plants

[0035] 1. GmABI3 gene sequence, SEQ ID NO.1:

[0036] ATGGAGTGTGAAGTTGAATTACAAGGGGGAGATCTGCATGCAGGGGAGGTAACT

[0037] GACCCAAACCCTATTGGTTTTGGCAACATGGAAGATGAACACACTTTGGCGGTTGCTG

[0038] AGAGAGAGATGTGGCTGAACAGTGACCAAGACGAGTTCCTAGGTGTCAATGATGCTT

[0039] CCATGTTCTACGCTGATTTCCCTCCTCTCCCTGATTTCCCTTGCATGTCATCATCATCAT

[0040] CTTCATCTTCAGCAACACCACTTCCCGTGAAGACCATGACATGTTCCACCACCACCAC

[0041] CACTTCCTCTTCTTCCTCTTCCTCTTCTTGGGCCATGTTGAAGTCAGATGCTGAGGAAG

[0042] ATGCAGAGAAAAACCATTGCAACCGATACATGCATGACCAACTTGATGCAACTTTGTC

[0043] TTCCACCGCTTCCATGGAGATTTCTCAACAGCAAAACCCTGATCCTGGCCTTGGTGGC

[0044] ACTGTTGGAGAGTGCATGGATGATGTTATGGACACTTTTGGTTACATGGAGCTTTTGG

[0045] AGGCCAATGATTTCTTTGACCCTGCCTCTATCTTTCAGAACGAGGGTAACGAAAACCC

[0046] TTTAGAGGAGTTTGGCACACTGGAGGAGCATGTGCCGTTTCATGAAGAGCAACATGC

[0047] AATGGTGCATCATCAGCAAGGGCAAGCAGAAGAGGAAGATCATCAGGTTCCTTTTTG

[0048] TGAAGAGATCCAAGGAGATGAAGAAGGTGGTGATGGTGTTGGAGTAGATGATGAGAT

[0049] GAGTAATGTGTTCTTGGAGTGGCTTAAGTCTAACAAGGATAGTGTCTCAGCTAATGAC

[0050] TTGAGGAATGTGAAGCTCAAGAAGGCGACACTCGAAAGCGCGGCGAGGCGGCTAGG

[0051] GGGAGGAAAAGAAGCCATGAAGCAGTTGCTGAAGCTGATTCTTGAGTGGGTTCAAAC

[0052] CAGCCATCTTCAGAATAAGCGCCGAAAGGAGAATAATGGTAGTAGTATTAGCAGTGT

[0053] ACTTCAGGCACAGTTTCAGGATCCTAGTGGCCAGAACAACAACCAGAATACACAAAG

[0054] TGGTTCATTTGCACCTGAATCAAACACTTGTTTCAACAACCAAACACCATGGTTGAGT

[0055] TCTCAAACTTTTGCAACAGATCAGGCTCCTCTCATGGTGCCTCCACAGCAATTTCCACA

[0056] ACCCATGGTTGGGTATGTGGGTGACCCTTACACTAGTGGTGCTGCTTCAAATAACATA

[0057] TCAGCCACTCATAACCATAACAACAGCAACCCTTATCAACCTGGTGCAGAACAATACC

[0058] ACATGTTGGAGTCAGCACATTCATGGCCACATTCTCTGTTCAATGTTGCTTCTAACTAT

[0059] AGTCAGTCTTTTGGGGACAATAATGGTCTTAACCCACATGGGGGTTTCGGTGGTGGAG

[0060] GCTATGGCAATAACCAGTACCCTTATCAGTTTTTTCATGGCCCTGGTGATAGGTTGATG

[0061] AGGTTGGGGCCCTCCGCGACGAAGGAAGCGAGGAAGAAGAGAATGGCAAGGCAAAG

[0062] AAGGTTTCTGTCTCATCACAGGCATCATAGTGGTAATCACCAGAATCAAGGGTCTGAC

[0063] CCTCATGCAAGAATGGGGGGTGATAATTGCAACACTGCTTTGGCTGCACCTCATCACG

[0064] CAAATCCTTCAGCCAATTGGATGTACTGGCAGGCTATGATTGGCGGCGCGGCAGGTCC

[0065] TTTGGCTCCGGTGATTCCGGCCGAGCCGCCGGTGGTACAACCGGTCGTGGACCGGTCG

[0066] GCCATGCAGACACAGAATTGTCATCAGAATCGAGTTGCATCAGATAGGAGACAGGGT

[0067] TGGAAGCCTGAGAAGAACTTGAGGTTCCTTCTGCAGAAGGTGTTGAAACAAAGCGAT

[0068] GTTGGAAGTTTGGGGAGAATAGTTTTGCCAAAAAAAGAGGCAGAAACCCATTTGCCA

[0069] GAGCTGGAGGCAAGAGATGGAATTTCCATAACAATGGAAGACATTGGAACTTCACGT

[0070] GTTTGGAACATGCGCTATAGCATCAGATACTGGCCGAACAACAAAAGCAGAATGTAT

[0071] TTGCTCGAGAACACTGGTGACTTTGTGAGAGCCAATGGACTCCAAGAGGGAGATTTCA

[0072] TAGTGATATACTCAGATGTGAAGTGTGGCAAATATATGATAAGAGGAGTGAAAGTGA

[0073] GGCAACAAGGTGTGAAACCAGAGACCAAGAAAGCAGGAAAATCGCAGAAAAACCAG

[0074] CATGGGACAGGGACTAATGCATCAAGTACAGCTGGTACTGCTGCTAATAATGGCACGTCATCGTCACCGAAAACCAAATCTGAAAAAAGTAGTAAATTAATATAA;

[0075] 2. Amino acid sequence of GmABI3, SEQ ID NO.2:

[0076] MECEVELQGGDLHAGEVTDPNPIGFGNMEDEHTLAVAEREMWLNSDQDEFLGVNDASM

[0077] FYADFPPLPDFPCMSSSSSSSSATPLPVKTMTCSTTTTTSSSSSSSSWAMLKSDAEEDAEKN

[0078] HCNRYMHDQLDATLSSTASMEISQQQNPDPGLGGTVGECMDDVMDTFGYMELLEANDF

[0079] FDPASIFQNEGNENPLEEFGTLEEHVPFHEEQHAMVHHQQGQAEEEDHQVPFCEEIQGDEE

[0080] GGDGVGVDDEMSNVFLEWLKSNKDSVSANDLRNVKLKKATLESAARRLGGGKEAMKQ

[0081] LLKLILEWVQTSHLQNKRRKENNGSSISSVLQAQFQDPSGQNNNQNTQSGSFAPESNTCFN

[0082] NQTPWLSSQTFATDQAPLMVPPQQFPQPMVGYVGDPYTSGAASNNISATHNHNNSNPYQ

[0083] PGAEQYHMLESAHSWPHSLFNVASNYSQSFGDNNGLNPHGGFGGGGYGNNQYPYQFFH

[0084] GPGDRLMRLGPSATKEARKKRMARQRRFLSHHRHHSGNHQNQGSDPHARMGGDNCNT

[0085] ALAAPHHANPSANWMYWQAMIGGAAGPLAPVIPAEPPVVQPVVDRSAMQTQNCHQNR

[0086] VASDRRQGWKPEKNLRFLLQKVLKQSDVGSLGRIVLPKKEAETHLPELEARDGISITMEDI

[0087] GTSRVWNMRYSIRYWPNNKSRMYLLENTGDFVRANGLQEGDFIVIYSDVKCGKYMIRGVKVRQQGVKPETKKAGKSQKNQHGTGTNASSTAGTAANNGTSSSPKTKSEKSSKLI;

[0088] 1) Construction of pGES201-GmABI3 knockout vector

[0089] The GmABI3-CDS sequence sgRNA target was designed by CRISPR-GE website. Finally, the CDS sequence 84-103

[0090] An optimal target sequence was determined at bp: GGAAGATGAACACACTTTGG (SEQ ID NO. 3), with a PAM site of CGG and a GC content of 45%. Next, the designed target sequence was constructed into the vector pGES201.

[0091] The pGES201 vector into which the SgRNA target sequence was transferred was detected by PCR specific cloning. Specific primers were designed upstream and downstream of the target site for testing. The upstream primer KOABI3-F: TGAGATACCCCTATCCCTTTGCT (SEQ ID NO. 4) and the downstream primer KOABI3-R: CATGCTCCTCCAGTGTGCCA (SEQ ID NO. 5). The results are shown in Figure 5. Figure 1 As shown: The test results ultimately detected the target sequence at 520bp. Combined with the company's sequencing results, it was preliminarily confirmed that the specific sgRNA target was successfully transferred into the pGES201 vector. Finally, genetic transformation was performed to detect target editing and protein expression changes.

[0092] 2) Genetic transformation of transgenic soybeans

[0093] The expression vector (pSOY1-GmABI3) and knockout vector (pGE S201-GmABI3) were transformed into the recipient variety Dongnong 50 using Agrobacterium-mediated soybean cotyledonary node transformation method. Figure 5 As shown: After explant infection, co-cultivation, cluster bud induction, cluster bud screening and elongation, rooting and seedling hardening, overexpression regenerated plants and mutant regenerated plants were obtained, which were used for subsequent transgenic plant propagation and data collection.

[0094] 3) Schematic diagram of Gmabi3 knockout target

[0095] T2 generation Gmabi3 mutant seeds were propagated in the seedling room, and leaf DNA was extracted. The edited soybean variety DN50 was used as a negative control. Detection primers were designed upstream and downstream of the gene editing site for PCR amplification, and the unpurified amplified products were sent to Sanger sequencing. Figure 6 As shown: Two types of mutations were detected in the GmABI3 gene sequence:

[0096] Gmabi3-1: Mutation type 1: At the target site 1, 27 bases (AACAGTGACCAAGACGAGTTCCTAGGT, SEQ ID NO. 16) were inserted at 105 bp of the CDS sequence, and one base (G) was inserted at 453 bp. The sequencing result was a single peak. After amino acid translation online, it was found that the translation of amino acid 170 was terminated, resulting in the loss of function of the B3 domain.

[0097] Gmabi3-2: Mutation type 2: At the position of target 2, a base (T) was inserted at 561bp of the CDS sequence. The sequencing result was a single peak. After amino acid translation online, it was found that the translation of amino acid 189 was terminated, resulting in the loss of function of the B3 domain.

[0098]

[0099]

[0100] Example 2. Construction of GmABI3 overexpression plants

[0101] 1) GmABI3 gene cloning

[0102] The CDS sequence of the GmABI3 gene was determined based on the soybean genome sequence in the Phytozome V13 comprehensive database. Specific primers for the candidate gene were designed using SnapGene software. Total RNA was extracted from leaves of soybean variety SN14 and reverse transcribed into cDNA, which was used as a template for PCR amplification. The upstream primer GmABI3-cF was: GGATCCATGGAGTGTGAAGT TGAATTACAA (SEQ ID NO. 6), and the downstream primer GmABI3-cR was: TCTAGATTATATTAATTTAC TACTTTTTTCAGATTTGG (SEQ ID NO. 7). Figure 2 As shown, after electrophoresis detection, an electrophoresis band consistent with the expected fragment size of 2286 bp was obtained, and the sequencing results were consistent with expectations.

[0103] 2) Construction of FU28-GmABI3 entry vector

[0104] The recovered target fragment and FU28 empty vector were double-digested with XbaI and BamHI restriction endonucleases, and then ligated with SolutionI. The ligated product was transformed into DH5α and cultured on a plate containing the corresponding resistance until a single colony grew. The single colony was identified by PCR using the GmABI3 specific primer and sent to Bio-Tech for sequencing. The results are as follows. Figure 3 As shown, the target band was detected in the positive control, but not in the negative control. The target fragments were detected at the 2286 bp position in all clones 1-8, indicating that the entry vector Fu28-GmABI3 was successfully constructed.

[0105] 3) Construction of pSOY1-GmABI3 expression vector

[0106] Extract the constructed entry vector (FU28-GmABI3) plasmid and the empty plasmid of the expression vector pSOY1 strain, obtain the recombinant plasmid through LR reaction and transform it into Escherichia coli DH5α, culture it on a plate containing the corresponding resistance until a single colony grows, use the single colony and GmABI3 specific primers to perform PCR identification and send it to Biotechnology for sequencing. After successful sequencing, transform it into EHA105 Agrobacterium, grow a single colony on the resistance plate, and after PCR identification, freeze the bacterial solution at -80℃. The results are as follows Figure 4 As shown: The expected target band was detected at 2286 bp by electrophoresis, indicating that the expression vector was constructed and used for subsequent experiments.

[0107] 4) Genetic transformation of transgenic soybeans

[0108] The expression vector (pSOY1-GmABI3) and knockout vector (pGE S201-GmABI3) were transformed into the recipient variety Dongnong 50 using Agrobacterium-mediated soybean cotyledonary node transformation method. Figure 5 As shown: After explant infection, co-cultivation, cluster bud induction, cluster bud screening and elongation, rooting and seedling hardening, overexpression regenerated plants and mutant regenerated plants were obtained, which were used for subsequent transgenic plant propagation and data collection.

[0109] 5) PCR detection of GmABI3 overexpressing plants

[0110] The seeds of the T3 generation overexpressing plants were sown in the phenotyping garden of Northeast Agricultural University. When the first trifoliate compound leaf unfolded, DNA from the leaves of the transgenic plants was extracted. Primers were designed and amplified based on the full length of the gene (CDS) and the full length of GFP on the vector fragment. The upstream primer ABI3gene-F: GTAACTGACCCAAACCCTAT (SEQ ID NO. 8) and the downstream primer GFP2-R: GTCCTTGAAGAAGATGGTGC (SEQ ID NO. 9) were used for amplification. Figure 7 As shown: a clear and single band can be detected at 2706bp, which is consistent with the size of the positive plasmid band. The wild type is used as a negative control, without the GFP fragment on the vector and the target band. Lanes 3-13 show that a total of 11 positive plants were obtained.

[0111] 6) qRT-PCR identification of GmABI3 overexpressing plants

[0112] qRT-PCR was used to analyze the expression level changes in transgenic plants and control plants. ABI3-qF: GATCCTAGTGGCCAGAACAACAACC (SEQ ID NO.10), ABI3-qR: CCAACATGTGGTATTGTTCT GCACC (SEQ ID NO.11) with GmActin4 as the internal reference gene, the upstream primer sequence was GmActin4-F: GTGTCAGCCATACTGTCCCCATTT (SEQ ID NO.12), GmActin4-R: GTTTCAAGCTCTTGC TCGTAATCA (SEQ ID NO.13) and the expression level of Dongnong 50 was used as a reference for qRT-PCR amplification. The results are shown in Figure 1. Figure 8 As shown, the expression levels of the three overexpression lines were significantly higher than those of the control plants, and there were extremely significant differences. The three high-expression lines were named GmABI3-1, GmABI3-2, and GmABI3-3 in future experiments.

[0113] 7) Western blot analysis of GmABI3 overexpressing plants

[0114] In order to further identify the positive overexpression plants, Western blot was used to detect at the protein level. The overexpression transgenic plants with consistent growth and robustness were selected, and the total protein of the leaf tissue was extracted for Western Blot detection. The results are shown in Figure 2. Figure 9 As shown, the target protein band was detected around 110.98 kDa in the leaf tissues of the three overexpression lines, and was consistent with the predicted size.

[0115] Example 3. Determination of quality traits of GmABI3 transgenic soybean grains

[0116] 1. Determination of protein and oil content in T3 and T4 generation transgenic soybean grains

[0117] The T2 generation mutant seeds (Gmabi3), control seeds (WT), and T1 generation overexpression seeds (GmABI3) harvested in the seedling room in 2021 will continue to be planted in the seedling room for another generation to T2, and will be planted in the potting field of Northeast Agricultural University in 2022. Under the same water and fertilizer conditions, after the seeds mature, the T3 generation seeds will be collected according to the above-mentioned test lines, and the harvested seeds will be used for subsequent phenotypic identification in 2023, and the total nitrogen content of soybean seeds will be measured by the Dumas nitrogen method.

[0118] The results are as follows Figure 10 As shown in A: In the T3 generation mutant seeds, the average total nitrogen content of Gmabi3-1 was 33.31%, that of Gmabi3-2 was 30.21%, and the protein content of the two lines was extremely significantly lower than that of WT plants. The average total nitrogen content of WT was 40.41%, the average total nitrogen content of overexpressed GmABI3-1 was 44.62%, GmABI3-2 was 43.91%, and GmABI3-3 was 43.35%, and the total nitrogen content of the three lines was extremely significantly higher than that of WT. The average total nitrogen content of T4 generation mutant seeds Gmabi3-1 was 37.73%, Gmabi3-2 was 43.91%, and GmABI3-3 was 43.35%. The total nitrogen protein content of the two lines was significantly lower than that of the WT plants. The average total nitrogen content of WT was 40.31%, the average total nitrogen content of the overexpressed GmABI3-1 was 42.59%, GmABI3-2 was 43.43%, and GmABI3-3 was 42.90%. The total nitrogen content of the three lines was significantly higher than that of WT.

[0119] The crude oil content of mature grains of T3 generation seeds was determined using a near infrared grain analyzer. Figure 10As shown in B: The average oil content of the mutant Gmabi3-1 was 23.57%, and the average oil content of Gmabi3-2 was 22.40%. The oil content of the two strains was significantly higher than that of the WT plants. The average oil content of WT was 20.93%, the average oil content of the overexpressed GmABI3-1 was 18.31%, GmABI3-2 was 18.88%, and GmABI3-3 was 18.52%. The average oil content of the three strains was significantly lower than that of WT. Using the Soxhlet extraction method, the T4 generation seeds showed that the average oil content of the mutant Gmabi3-1 was 22.17%, and the average oil content of Gmabi3-2 was 22.53%. The oil content of both strains was significantly higher than that of WT. The average oil content of the overexpressing GmABI3-1 was 18.53%, GmABI3-2 was 18.63%, and GmABI3-3 was 18.30%. The average oil content of the three strains was significantly lower than that of WT, and the phenotypic trends of the two years were consistent.

[0120] The results are as follows Figure 10 As shown in Figure C, the palmitic acid, linoleic acid, and linolenic acid contents of mutants Gmabi3-1 and Gmabi3-2 were significantly higher than those of WT plants, while stearic acid was significantly higher than that of WT plants, and oleic acid was significantly lower than that of WT plants. The total fatty acid content of mutants was significantly higher than that of WT plants. The linolenic acid content of overexpressed GmABI3-1, GmABI3-2, and GmABI3-3 was significantly higher than that of WT plants, while the stearic acid content of GmABI3-1, GmABI3-2, and GmABI3-3 did not change significantly. It is worth noting that the oleic acid content of GmABI3-1, GmABI3-2, and GmABI3-3 was significantly lower than that of WT plants.

[0121] 2. Transmission electron microscopy observation of T4 transgenic soybeans

[0122] Seeds of DN50, mutant Gmabi3-1, and overexpressed GmABI3-2 grown under the same environment at the MM stage (50-80 days after flowering) were observed by transmission electron microscopy. Figure 11 As shown: within the range of 5μm, it was found that the number of oil bodies of the mutant Gmabi3-1 was extremely significantly higher than that of WT, and the number of protein bodies was extremely significantly lower than that of WT; the number of protein bodies of the overexpressed GmABI3-2 was extremely significantly higher than that of WT.

[0123] 3. Observation of agronomic traits of T4 transgenic soybeans

[0124] In 2023, the mutant (Gmabi3), overexpression (GmABI3) and control (WT) plants were grown in the same environment at the potting farm of Northeast Agricultural University. Samples were taken after the plants were fully mature to investigate the agronomic traits of plant height. Figure 12As shown in the figure, compared with the WT control plants, the two mutant lines showed significantly lower plant heights, while the three overexpression lines showed significantly higher plant heights. Loss of GmABI3 gene function can increase the content of various fatty acid components in the grain and reduce the total nitrogen content. Therefore, it can be inferred that the GmABI3 gene may negatively regulate oil content in soybean grains and positively regulate protein content.

Claims

1. Application of overexpressing GmABI3 protein in reducing oil content in soybean, characterized in that: The sequence of the GmABI3 protein is shown in SEQ ID NO.

2.

2. Application of overexpressing GmABI3 gene in reducing oil content in soybean, characterized in that: The sequence of the GmABI3 gene is shown in SEQ ID NO.

1.

3. Use of a vector containing an overexpressed GmABI3 gene in reducing soybean oil content, characterized in that: The sequence of the GmABI3 gene is shown in SEQ ID NO.

1.

4. Use of a recombinant microbial cell containing an overexpressed GmABI3 gene in reducing soybean oil content, characterized in that: The sequence of the GmABI3 gene is shown in SEQ ID NO.

1.

5. A method for increasing soybean oil content, characterized in that: The steps of the method are as follows: (1) inserting the amplified sgRNA sequence into a vector; the sgRNA sequence is shown in SEQ ID NO.3; (2) introducing the vector obtained in step (1) into Agrobacterium, and using Agrobacterium to transfer the vector into soybean to obtain transgenic soybean; (3) Identifying the transgenic soybeans obtained in step (2) to obtain positive transgenic soybeans.

6. Use of soybean plants containing knocked-out GmABI3 gene for increasing soybean oil content, characterized in that: The sequence of the GmABI3 gene is shown in SEQ ID NO.1.

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