Method for improving soybean seed cotyledon color and grain quality by gene editing GmTT8 gene and application thereof

Abstract

The present application relates to the technical field of soybean breeding, and particularly relates to an sgRNA combination targeting a soybean GmTT8 gene and application thereof. The present application uses the sgRNA combination targeting the soybean GmTT8 gene to perform site-directed editing on the soybean GmTT8 gene, and obtains a GmTT8 gene edited soybean material. Compared with the wild type, the edited material exhibits phenotypes such as lighter seed hilum color, reduced seed hilum area and seed hilum length, increased grain protein content, reduced grain oil content, increased grain size, and increased protein-lipid ratio. It is shown that the GmTT8 gene is involved in the formation of soybean seed hilum color and the regulation of grain quality traits, and can be used as a gene editing target point for improving soybean seed hilum color and grain quality. The present application provides a new gene resource and technical approach for creating new soybean germplasm with high protein, high protein-lipid ratio and light seed hilum color.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to a combination of sgRNAs targeting the soybean GmTT8 gene and their applications. Background Technology

[0002] Soybean (Glycinemax) is a globally important food crop, oilseed crop, and source of plant protein. Its seeds are rich in protein, oil, fatty acids, amino acids, and various functional metabolites, making them a crucial raw material for plant protein foods, edible oils, and feed processing. Soybean seed quality encompasses not only nutritional traits such as protein content, oil content, fatty acid composition, and amino acid composition, but also appearance traits such as seed coat color, hilum color, and seed size. These traits collectively influence the nutritional, processing, and commercial value of soybeans. With the increasing demand for high-quality, specialized soybean breeding, cultivating new soybean germplasm with high protein, high protein-to-fat ratio, and excellent appearance has become an important direction for soybean quality improvement. However, soybean seed quality and appearance traits are influenced by genetic background, developmental process, and environmental conditions. Traditional breeding methods suffer from long cycles, low efficiency, and insufficient stability of target traits in the synergistic improvement of multiple quality traits. Therefore, identifying key genes regulating soybean seed quality and appearance traits and using gene editing technology for targeted improvement is of great significance for creating high-quality soybean germplasm resources and promoting molecular design breeding of soybeans.

[0003] Hilum color in soybeans is a crucial component of soybean seed appearance quality and a key trait for variety identification, seed phenotypic assessment, and commercial evaluation. Significant differences exist in the seed coat and hilum colors among different soybean materials, and their formation is closely related to the synthesis, translocation, and accumulation of pigments in the seed coat or hilum. Existing research indicates that soybean seed coat color is primarily regulated by classical genetic loci such as I, R, and T, and its formation is closely related to the synthesis and accumulation of anthocyanins, proanthocyanidins, and other flavonoids. Differences in anthocyanin, isoflavone, total phenols, total flavonoids, as well as seed components such as protein, oil, and fatty acids, exist among soybean materials with different seed coat colors, suggesting that pigment metabolism pathways may be associated with seed development and the accumulation of stored substances. Therefore, identifying key genes involved in both soybean hilum color formation and seed quality regulation is of great significance for creating new soybean germplasm with synergistic improvements in both appearance and nutritional quality.

[0004] TT8 is an important bHLH transcription factor in plants, belonging to the key regulatory factors in the flavonoid metabolism regulatory network. Previous studies have shown that TT8 can form the MYB-bHLH-WD40 transcriptional regulatory complex with MYB-type transcription factors and WD40 protein, regulating the expression of downstream structural genes in the flavonoid metabolic pathway, participating in the biosynthesis of pigments such as anthocyanins and proanthocyanidins, and playing an important role in seed coat pigment deposition, transparent seed coat phenotype formation, and seed development. Simultaneously, TT8-type transcription factors may also participate in seed fatty acid synthesis, embryonic development, and the accumulation of storage substances. The soybean genome contains the TT8 homolog GmTT8, which may be involved in processes related to soybean hilum pigment accumulation and grain development. However, systematic research on the role of the GmTT8 gene in soybean hilum color formation, grain protein content, oil content, grain size, and protein-to-fat ratio remains lacking, especially in terms of technical solutions for targeted modification of GmTT8 using gene editing technology for soybean quality improvement.

[0005] Current soybean quality improvement technologies mostly focus on improving single traits such as protein content, oil content, fatty acid composition, or stress resistance, while gene resources and editing targets for the synergistic regulation of hilum color and grain quality traits are relatively insufficient. In particular, existing technologies lack an effective method to selectively edit the GmTT8 gene to lighten soybean hilum color, reduce hilum area and length, increase grain protein content, reduce oil content, increase grain size, and improve the protein-to-fat ratio. Therefore, it is necessary to provide a new soybean quality improvement target based on the GmTT8 gene and its application method to meet the needs of high-quality soybean germplasm creation and molecular design breeding. Summary of the Invention

[0006] To address the shortcomings in existing technologies, such as insufficient discovery of genes regulating soybean seed quality and a limited number of molecular targets for improving soybean seed quality, this invention aims to identify important genes controlling soybean seed quality and utilize gene editing techniques to alter soybean seed quality. Therefore, this invention provides a combination of sgRNAs targeting the soybean GmTT8 gene and its applications, particularly in regulating hilum color and soybean seed quality.

[0007] The first objective of this invention is to provide the application of the soybean GmTT8 gene in regulating soybean grain quality.

[0008] Preferably, the GmTT8 gene is a homologous gene of soybean TT8, encoding a bHLH-type transcription factor.

[0009] More preferably, the GmTT8 gene is Glyma.02G147800, and its CDS nucleotide sequence is shown in SEQ ID NO.1.

[0010] A second objective of this invention is to provide the application of gene editing of the GmTT8 gene in improving soybean seed quality. Preferably, the improved soybean seed quality comprises at least one of the following:

[0011] (1) Regulate the protein content of soybean seeds;

[0012] (2) Regulate the oil content of soybean seeds;

[0013] (3) Regulate the egg-to-fat ratio of soybean seeds;

[0014] (4) Regulate soybean seed size or shape; (5) Regulate soybean hilum color.

[0015] More preferably, the gene-edited GmTT8 gene is used to target and edit the coding region of the soybean GmTT8 gene, causing base insertion, deletion, or substitution mutations in the GmTT8 gene, thereby obtaining GmTT8 gene-edited soybean materials. Compared with the wild type, the GmTT8 gene-edited soybean materials exhibit lighter hilum color, reduced hilum area and / or hilum length, increased seed protein content, reduced oil content, increased egg-to-fat ratio, and / or larger seed size.

[0016] To achieve targeted editing of the soybean GmTT8 gene, this invention further provides a combination of sgRNAs targeting the soybean GmTT8 gene. The sgRNA combination includes a first sgRNA and a second sgRNA; wherein the first sgRNA targets a first target site in the coding region of the GmTT8 gene, and the second sgRNA targets a second target site in the coding region of the GmTT8 gene. The recognition sequence of the first sgRNA is 5′-ATGACTGCGCCACTAGACACTGG-3′, where the TGG at the 3′ end is a PAM sequence; the recognition sequence of the second sgRNA is 5′-AGACAAATCCGCCGACTCGCCGG-3′, where the CGG at the 3′ end is a PAM sequence.

[0017] This invention also provides a GmTT8 dual-target gene editing vector containing the aforementioned sgRNA combination. The gene editing vector includes an sgRNA expression module, a Cas9 nuclease expression module, and a plant selection marker expression module. The sgRNA expression module is an AtU6-26p promoter-driven tRNA-sgRNA tandem expression module, where the first sgRNA and the second sgRNA exist in tandem on the vector. The tRNA-sgRNA tandem expression module sequentially includes a tRNA sequence, a first sgRNA recognition sequence, an sgRNA backbone sequence, a tRNA sequence, a second sgRNA recognition sequence, and an sgRNA backbone sequence.

[0018] This invention also provides an Agrobacterium engineered strain containing the GmTT8 dual-target gene editing vector. Using this Agrobacterium engineered strain to genetically transform soybean recipient material, GmTT8 gene-edited soybean material can be obtained.

[0019] A third objective of this invention is to provide a method for improving soybean seed quality, comprising the step of gene editing of the soybean GmTT8 gene.

[0020] Preferably, the gene editing is a site-specific editing of the CDS region of the GmTT8 gene.

[0021] More preferably, the gene editing causes base insertion, deletion, or substitution in the GmTT8 gene to obtain GmTT8 gene-edited soybean material.

[0022] The fourth objective of this invention is to provide a GmTT8 gene-edited soybean material.

[0023] Preferably, in the GmTT8 gene-edited soybean material, the GmTT8 gene undergoes an editing mutation.

[0024] More preferably, the GmTT8 gene-edited soybean material exhibits at least one of the following traits:

[0025] (1) Increased protein content in grains;

[0026] (2) The oil content of the seeds decreased;

[0027] (3) The egg-to-fat ratio of the grain increased;

[0028] (4) The color of the hilum becomes lighter;

[0029] (5) The size or shape of the grains has changed.

[0030] Specifically, the present invention provides the following technical solution:

[0031] In a first aspect, the present invention provides a combination of sgRNAs targeting the soybean GmTT8 gene, the sgRNA combination comprising a first sgRNA and a second sgRNA targeting the soybean GmTT8 gene; the first sgRNA and the second sgRNA respectively target different sites in the coding region of the soybean GmTT8 gene; preferably, the recognition sequence of the first sgRNA is shown in SEQ ID NO.4, and the recognition sequence of the second sgRNA is shown in SEQ ID NO.5, wherein the 3′ TGG of SEQ ID NO.4 and the 3′ CGG of SEQ ID NO.5 are respectively the PAM sequences required for Cas9 recognition; more preferably, the amino acid sequence of the protein encoded by the GmTT8 gene is shown in SEQ ID NO.3; more preferably, the nucleotide sequence of the GmTT8 gene is shown in SEQ ID NO.1; and even more preferably, the coding sequence of the GmTT8 gene is shown in SEQ ID NO.2.

[0032] Secondly, the present invention provides a gene editing vector targeting the soybean GmTT8 gene, wherein the gene editing vector contains the sgRNA combination as described in claim 1.

[0033] As described above, the T-DNA region of the gene editing vector includes a right boundary RB, an sgRNA expression module, a Cas9 nuclease expression module, a plant selection marker expression module, and a left boundary LB; wherein, the sgRNA expression module is used to express a first sgRNA and a second sgRNA targeting different sites of the GmTT8 gene.

[0034] As described above, in the gene editing vector, the first sgRNA and the second sgRNA exist in the gene editing vector as a tRNA-sgRNA tandem expression module driven by the AtU6-26p promoter; the tRNA-sgRNA tandem expression module includes a tRNA sequence, a first sgRNA recognition sequence, a second sgRNA recognition sequence, and an sgRNA backbone sequence.

[0035] As described above, the gene editing vector includes a 35S promoter, a Cas9 nuclease coding sequence, and a Tnos terminator; preferably, the Cas9 nuclease expression module includes a 35S promoter, a 3×FLAG tag sequence, a nuclear localization signal NLS, a bdCas9 coding sequence, a nuclear localization signal NLS, and a Tnos terminator.

[0036] As described above, the plant selection marker expression module of the gene editing vector includes a 35S promoter, a Bar selection marker gene, and a poly(A) termination sequence.

[0037] Thirdly, the present invention provides a biological material containing the sgRNA combination of claim 1 or the gene editing vector of any one of claims 2-6, or obtained by gene editing using the sgRNA combination of claim 1 or the gene editing vector of any one of claims 2-6. The biological material is an expression cassette, transposon, plasmid vector, viral vector, engineered bacteria, or transgenic cell line, preferably Agrobacterium engineered bacteria. Preferably, the biological material obtained by gene editing using the sgRNA combination of claim 1 or the gene editing vector of any one of claims 2-6, relative to the wild type, has a TAGAC deletion mutation in the recognition sequence of the first sgRNA as shown in SEQ ID NO. 4, and the corresponding sequence after mutation is ATGACTGCGCCACACTGG.

[0038] Fourthly, the present invention provides a method for improving soybean quality using gene editing, comprising the following steps: introducing the sgRNA combination of claim 1, the gene editing vector of any one of claims 2-6, or the biological material of claim 7 into soybean recipient material, so that the sgRNA combination of claim 1 guides Cas nuclease to perform site-specific editing of the soybean GmTT8 gene, thereby obtaining GmTT8 gene-edited soybean material;

[0039] Preferably, the site-specific editing of the soybean GmTT8 gene includes generating a base insertion, deletion, or substitution mutation at the target site of the GmTT8 gene, wherein the mutation causes a change in the coding sequence of the GmTT8 gene;

[0040] Preferably, the result of the site-directed editing of the soybean GmTT8 gene is that, relative to the wild type, a TAGAC deletion mutation is generated in the recognition sequence of the first sgRNA as shown in SEQ ID NO.4, and the corresponding sequence after mutation is ATGACTGCGCCACACTGG.

[0041] Preferably, the improved soybean seed quality comprises at least one of the following:

[0042] (1) Regulate the protein content of soybean seeds;

[0043] (2) Regulate the oil content of soybean seeds;

[0044] (3) Regulate the egg-to-fat ratio of soybean seeds;

[0045] (4) Regulate soybean seed size or shape;

[0046] (5) Regulate the color of the soybean hilum.

[0047] Fifthly, this invention provides the application of the soybean GmTT8 gene as a gene editing target in improving soybean quality.

[0048] The improved soybean seed quality includes at least one of the following:

[0049] (1) Regulate the protein content of soybean seeds;

[0050] (2) Regulate the oil content of soybean seeds;

[0051] (3) Regulate the egg-to-fat ratio of soybean seeds;

[0052] (4) Regulate soybean seed size or shape;

[0053] (5) Regulating the color of the soybean hilum;

[0054] Preferably, SEQ ID NO.4 and SEQ ID NO.5 in the soybean GmTT8 gene are used as gene editing targets.

[0055] Beneficial Effects: This invention utilizes gene editing technology to perform site-specific editing of the soybean GmTT8 gene, obtaining GmTT8 gene-edited soybean materials. Compared to the wild type, the edited materials exhibit significantly lighter hilum color, increased seed protein content, decreased oil content, and increased protein-to-fat ratio, indicating that the GmTT8 gene plays a crucial role in the formation of soybean hilum color and the regulation of seed quality. This invention reveals the regulatory effect of GmTT8 gene editing on soybean seed quality and appearance, filling the application gap in improving soybean seed quality using GmTT8 gene editing. It provides potential gene resources and technical support for cultivating high-protein, light-hilum-colored, and other high-quality soybean materials, and has significant application value for promoting molecular breeding of soybean quality. Attached Figure Description

[0056] Figure 1 This is a schematic diagram of the T-DNA region structure of a dual-target gene editing vector targeting the soybean GmTT8 gene. The T-DNA region includes a right boundary RB, an AtU6-26p promoter-driven tRNA-sgRNA tandem expression module, a 35S promoter-driven Cas9 nuclease expression module, a plant selection marker bar expression module, a poly(A) termination sequence, and a left boundary LB. The tRNA-sgRNA tandem expression module contains two sgRNA recognition sequences, GmTT8-gRNA1 and GmTT8-gRNA2; the Cas9 nuclease expression module contains 3×FLAG, NLS, bdCas9, and NLS elements.

[0057] Figure 2 This is a schematic diagram of the structure of the soybean GmTT8 gene and its gene editing target sites; the boxes represent exons, the lines represent introns, and the arrows represent gene editing target sites.

[0058] Figure 3 This is a diagram showing the sequencing alignment results of the target site of the GmTT8 gene-edited strain; WT is the wild type, and 23-6 is the T1 generation 6 GmTT8 gene-edited strain.

[0059] Figure 4 The image shows the hilum color phenotypic diagram of mature seeds of wild-type soybean and the No. 6 GmTT8 gene-edited line; WT represents wild-type soybean, and 23-6 represents the T1 generation No. 6 GmTT8 gene-edited line.

[0060] Figure 5 This is a statistical graph showing the hilum phenotype of wild-type soybean and GmTT8 gene-edited lines; A represents hilum length, B represents hilum area; WT represents wild-type, and 23-6 represents the T1 generation 6 GmTT8 gene-edited line; data are expressed as mean ± standard error, and statistical analysis was performed using a two-tailed unpaired t-test. P < 0.001.

[0061] Figure 6 This is a statistical chart showing the seed quality indicators of wild-type soybean and GmTT8 gene-edited lines; A represents seed oil content, B represents seed protein content, and C represents the protein / oil ratio; WT represents wild-type, and 23-6 represents the T1 generation 6 GmTT8 gene-edited line; data are expressed as mean ± standard error, and statistical analysis was performed using a two-tailed unpaired t-test. P < 0.001. Detailed Implementation

[0062] The present invention will be further described below with reference to the embodiments, but the scope of protection of the present invention is not limited to the following embodiments. All equivalent substitutions or conventional improvements made based on the technical concept of the present invention should be included within the scope of protection of the present invention.

[0063] Example 1: Design of soybean GmTT8 gene editing target and construction of editing vector

[0064] 1.1 Identification of the GmTT8 gene

[0065] This embodiment uses the soybean GmTT8 gene as the target gene. The present invention has found that the GmTT8 gene is a gene in soybean encoding bHLH-type transcription factors. These transcription factors participate in the regulation of pigment metabolism pathways such as plant flavonoids, anthocyanins, and proanthocyanidins, and are related to pigment deposition and color formation in the seed coat or hilum.

[0066] Based on the nucleotide and coding sequences of the GmTT8 gene in the soybean reference genome, its coding region was analyzed to identify the GmTT8 gene as the gene editing target gene for regulating soybean hilum color and seed quality in this invention. The nucleotide sequence of the GmTT8 gene is shown in SEQ ID NO.1, the coding sequence of the GmTT8 gene is shown in SEQ ID NO.2, and the amino acid sequence of its encoded protein is shown in SEQ ID NO.3.

[0067] 1.2 Design of GmTT8 gene editing target and acquisition of sgRNA

[0068] Based on the coding sequence of the soybean GmTT8 gene, target site analysis and screening were performed on the coding region of the GmTT8 gene. Sites located in the exon region of the GmTT8 gene with adjacent sequences containing PAM sequences conforming to the 5′-NGG-3′ characteristic were selected as candidate editing targets, where N represents any base from A, T, C, or G. The selection criteria for target sites were: the target site being located in the coding region; the adjacent sequence containing the PAM sequence required for Cas9 nuclease recognition; high target sequence specificity; and suitability for gene editing vector construction.

[0069] After screening, two sgRNA recognition sequences targeting the coding region of the soybean GmTT8 gene were obtained, named GmTT8-gRNA1 and GmTT8-gRNA2, respectively. The sgRNA recognition sequence of GmTT8-gRNA1 is 5′-ATGACTGCGCCACTAGACACTGG-3′ (SEQ ID NO.4), where the 3′ TGG is a PAM sequence; the sgRNA recognition sequence of GmTT8-gRNA2 is 5′-AGACAAATCCGCCGACTCGCCGG-3′ (SEQ ID NO.5), where the 3′ CGG is a PAM sequence. The relative positions of the two sgRNA targets in the GmTT8 gene are as follows: Figure 2 As shown.

[0070] Both sgRNA recognition sequences are located in the coding region of the GmTT8 gene, and can guide the Cas9 nuclease to target different locations in the GmTT8 gene for site-specific cleavage. After repair of endogenous DNA in soybean cells, base insertion, deletion, or substitution mutations can be generated near the GmTT8 gene target site, thereby obtaining soybean gene-edited materials with altered GmTT8 gene function.

[0071] 1.3 Construction of GmTT8 dual-target gene editing vector

[0072] Based on the sgRNA recognition sequences of GmTT8-gRNA1 and GmTT8-gRNA2 obtained in section 1.2, a GmTT8 dual-target sgRNA expression module was constructed. This GmTT8 dual-target sgRNA expression module is an tRNA-sgRNA tandem expression module driven by the AtU6-26p promoter.

[0073] The tRNA-sgRNA tandem expression module is located downstream of the AtU6-26p promoter and includes a tRNA sequence, a GmTT8-gRNA1 recognition sequence, a GmTT8-gRNA2 recognition sequence, and an sgRNA backbone sequence. GmTT8-gRNA1 and GmTT8-gRNA2 exist in tandem within the same tRNA-sgRNA expression module, enabling the generation of sgRNAs targeting two different sites on the GmTT8 gene in plant cells.

[0074] The tRNA-sgRNA tandem expression module exists on the vector in the form of: AtU6-26p promoter—tRNA-sgRNA tandem expression module (GmTT8-gRNA1 / GmTT8-gRNA2).

[0075] After transcription in plant cells, this tRNA-sgRNA tandem expression module can generate sgRNAs targeting different sites of the GmTT8 gene via the endogenous tRNA processing system, which in turn guide the Cas9 nuclease to target two different target sites in the coding region of the GmTT8 gene.

[0076] The tRNA-sgRNA tandem expression module containing GmTT8-gRNA1 and GmTT8-gRNA2 was constructed into a plant CRISPR / Cas9 gene editing vector to obtain a GmTT8 dual-target gene editing vector that simultaneously targets two sites of the GmTT8 gene. The T-DNA region of the GmTT8 dual-target gene editing vector includes a right boundary RB, an AtU6-26p promoter, a tRNA-sgRNA tandem expression module, a 35S promoter, a Cas9 nuclease expression module, a Tnos terminator, a 35S promoter, a Bar selection marker gene, a poly(A) termination sequence, and a left boundary LB, as shown in the diagram. Figure 1 As shown.

[0077] The Cas9 nuclease expression module includes a 35S promoter, a 3×FLAG tag sequence, a nuclear localization signal NLS, a bdCas9 coding sequence, a nuclear localization signal NLS, and a Tnos terminator; the plant selection marker expression module includes a 35S promoter, a Bar selection marker gene, and a poly(A) termination sequence.

[0078] After soybeans are transformed with this GmTT8 dual-target gene editing vector, the GmTT8-gRNA1 and GmTT8-gRNA2 can respectively guide the Cas9 nuclease to target different sites of the GmTT8 gene, thereby generating mutations near the GmTT8 gene target sites.

[0079] 1.4 Validation of recombinant editing vectors and Agrobacterium-mediated transformation

[0080] The ligation product was transformed into competent E. coli cells, and positive single clones were obtained through resistance selection. The constructed GmTT8 dual-target gene editing vector was validated by PCR and sequencing, confirming that GmTT8-gRNA1 and GmTT8-gRNA2 were present in the expected order within the tRNA-sgRNA tandem expression module driven by the AtU6-26p promoter, and that the recognition sequences of the two sgRNAs were consistent with the designed sequences.

[0081] The validated GmTT8 dual-target gene editing vector was transformed into Agrobacterium strain EHA105. After resistance selection and PCR detection, Agrobacterium engineered strains containing the GmTT8 dual-target gene editing vector were obtained. These Agrobacterium engineered strains were used for subsequent soybean genetic transformation to obtain gene-edited soybean materials targeting the GmTT8 gene.

[0082] Example 2: Obtaining and molecularly identifying the GmTT8 gene-edited line No. 6

[0083] Using black-navel soybean as the recipient material, the Agrobacterium-mediated genetic transformation method was employed to introduce the GmTT8 gene-editing vector obtained in Example 1 into the soybean recipient material. After infection, co-culture, screening, and cultivation, candidate positive plants were obtained, and seeds of their self-pollinated progeny were harvested to obtain T1 generation GmTT8 gene-edited material.

[0084] Genomic DNA was extracted from young leaves of T1 generation candidate edited plants. Specific primers were designed based on the sequences flanking the GmTT8 gene target site to perform PCR amplification of the target region. The PCR amplification products were sequenced and compared with the wild-type GmTT8 gene sequence to identify the mutation type at the target site.

[0085] Sequencing alignment results showed that the T1 generation GmTT8 gene-edited strain No. 6 exhibited a sequence variation near the GmTT8 gene target site. This sequence variation altered the coding sequence of the GmTT8 gene, indicating that strain No. 6 was a positive material for GmTT8 gene editing. The sequencing alignment results of the target site of strain No. 6 are as follows: Figure 3 As shown.

[0086] Example 3: Identification of hilum color and morphology in GmTT8 gene-edited strain No. 6

[0087] Mature seeds of wild-type soybean and the No. 6 T1 generation GmTT8 gene-edited line were harvested. The mature seeds were photographed under the same light, background and shooting conditions to compare the hilum color phenotype of wild-type and No. 6 edited line.

[0088] The results showed that compared with the wild-type black hilum, the mature seeds of the GmTT8 gene-edited line 6 had a significantly lighter hilum color, exhibiting a light brown or diluted phenotype, indicating that GmTT8 gene editing can affect soybean hilum color formation. The comparison results of mature seed hilum color between the wild-type and the edited line 6 are shown below. Figure 4 As shown.

[0089] Further measurements of hilum length and area were performed on mature seeds of wild-type soybean WT and the GmTT8 gene-edited line 23-6 (No. 6). The hilum length measurement results showed ( Figure 5 In section A), the average hilum length of wild-type soybean WT was 1.98±0.11 mm, while that of the GmTT8 gene-edited line 23-6 (line 6) was 1.31±0.11 mm. Compared with wild-type soybean WT, the hilum length of the GmTT8 gene-edited line 23-6 was significantly reduced, by 33.7%. Hilum area measurements showed ( Figure 5 In the B category, the average hilum area of ​​wild-type soybean (WT) was 1.01 ± 0.08 mm. 2 The average hilum area of ​​the GmTT8 gene-edited line 23-6 (line 6) was 0.58 ± 0.07 mm. 2 Compared with wild-type soybean WT, the hilum area of ​​the GmTT8 gene-edited line 23-6 was significantly reduced, by 42.8%. Data are expressed as mean ± standard deviation. Statistical analysis was performed using a two-tailed unpaired t-test, and all differences reached a highly significant level (mean ± standard deviation). (P < 0.001). The results showed that editing the GmTT8 gene significantly reduced the hilum length and hilum area of ​​mature soybean grains.

[0090] Example 4: Determination of grain quality indicators of GmTT8 gene-edited strain No. 6

[0091] To investigate the effects of the GmTT8 gene on soybean seed quality traits, the oil content, protein content, and protein / oil ratio of mature seeds from wild-type soybean WT and the GmTT8 gene-edited line 23-6 (No. 6) were measured.

[0092] The results of the oil content determination showed that ( Figure 6In the A section, the average oil content of mature wild-type soybean WT seeds was 24.53±0.34%, while the average oil content of mature seeds of the 6 GmTT8 gene-edited line 23-6 was 21.34±0.46%. Compared with wild-type soybean WT, the oil content of mature seeds of the 6 GmTT8 gene-edited line 23-6 was significantly reduced, with a decrease of 13.0%.

[0093] The protein content determination results showed that ( Figure 6 In the study of wild-type soybean (WT), the average protein content of mature seeds was 35.27±0.76%, while the average protein content of mature seeds of the 6 GmTT8 gene-edited line 23-6 was 41.40±0.50%. Compared with wild-type soybean (WT), the protein content of mature seeds of the 6 GmTT8 gene-edited line 23-6 was significantly higher, with an increase of 17.4%.

[0094] The protein / oil ratio determination results showed that ( Figure 6 In the study of wild-type soybean WT, the average protein / oil ratio of mature seeds was 1.44±0.05, while that of the 6 GmTT8 gene-edited line 23-6 was 1.94±0.05. Compared with wild-type soybean WT, the protein / oil ratio of mature seeds of the 6 GmTT8 gene-edited line 23-6 was significantly higher, with an increase of 35.0%.

[0095] The above data are expressed as mean ± standard deviation. Each group had 6 replicates. Statistical analysis was performed using a two-tailed unpaired t-test. All differences in the indicators reached a highly significant level. P < 0.001. The results showed that GmTT8 gene editing could significantly reduce the oil content of mature soybean seeds, increase the protein content and protein / oil ratio, and thus change the oil and protein composition of soybean seeds.

Claims

1. A combination of sgRNAs targeting the soybean GmTT8 gene, characterized in that, The sgRNA combination includes a first sgRNA and a second sgRNA targeting the soybean GmTT8 gene; the first sgRNA and the second sgRNA target different sites in the coding region of the soybean GmTT8 gene, respectively; preferably, the recognition sequence of the first sgRNA is shown in SEQ ID NO.4, and the recognition sequence of the second sgRNA is shown in SEQ ID NO.5, wherein the 3′ TGG of SEQ ID NO.4 and the 3′ CGG of SEQ ID NO.5 are the PAM sequences required for Cas9 recognition, respectively; more preferably, the amino acid sequence of the protein encoded by the GmTT8 gene is shown in SEQ ID NO.3; more preferably, the nucleotide sequence of the GmTT8 gene is shown in SEQ ID NO.1; and even more preferably, the coding sequence of the GmTT8 gene is shown in SEQ ID NO.

2.

2. A gene editing vector targeting the soybean GmTT8 gene, characterized in that, The gene editing vector contains the sgRNA combination as described in claim 1.

3. The gene editing vector according to claim 2, characterized in that, The T-DNA region of the gene editing vector includes a right boundary RB, an sgRNA expression module, a Cas9 nuclease expression module, a plant selection marker expression module, and a left boundary LB; wherein, the sgRNA expression module is used to express a first sgRNA and a second sgRNA targeting different sites of the GmTT8 gene.

4. The gene editing vector according to claim 3, characterized in that, The first sgRNA and the second sgRNA exist in the gene editing vector as a tRNA-sgRNA tandem expression module driven by the AtU6-26p promoter; the tRNA-sgRNA tandem expression module includes a tRNA sequence, a first sgRNA recognition sequence, a second sgRNA recognition sequence, and an sgRNA backbone sequence.

5. The gene editing vector according to claim 3 or 4, characterized in that, The Cas9 nuclease expression module includes a 35S promoter, a Cas9 nuclease coding sequence, and a Tnos terminator; preferably, the Cas9 nuclease expression module includes a 35S promoter, a 3×FLAG tag sequence, a nuclear localization signal NLS, a bdCas9 coding sequence, a nuclear localization signal NLS, and a Tnos terminator.

6. The gene editing vector according to any one of claims 3-5, characterized in that, The plant selection marker expression module includes a 35S promoter, a Bar selection marker gene, and a poly(A) termination sequence.

7. A biomaterial, characterized in that, The biological material contains the sgRNA combination of claim 1 or the gene editing vector of any one of claims 2-6, or is obtained by gene editing using the sgRNA combination of claim 1 or the gene editing vector of any one of claims 2-6. The biological material is an expression cassette, transposon, plasmid vector, viral vector, engineered bacteria, or transgenic cell line, preferably Agrobacterium engineered bacteria. Preferably, the biological material obtained by gene editing using the sgRNA combination of claim 1 or the gene editing vector of any one of claims 2-6, relative to the wild type, has a TAGAC deletion mutation in the recognition sequence of the first sgRNA as shown in SEQ ID NO. 4, and the corresponding sequence after mutation is ATGACTGCGCCACACTGG.

8. A method for improving soybean quality using gene editing, characterized in that, The process includes the following steps: introducing the sgRNA combination of claim 1, the gene editing vector of any one of claims 2-6, or the biological material of claim 7 into soybean recipient material, so that the sgRNA combination of claim 1 guides the Cas nuclease to perform site-specific editing of the soybean GmTT8 gene, thereby obtaining GmTT8 gene-edited soybean material; Preferably, the site-specific editing of the soybean GmTT8 gene includes generating a base insertion, deletion, or substitution mutation at the target site of the GmTT8 gene, wherein the mutation causes a change in the coding sequence of the GmTT8 gene; Preferably, the result of the site-directed editing of the soybean GmTT8 gene is that, relative to the wild type, a TAGAC deletion mutation is generated in the recognition sequence of the first sgRNA as shown in SEQ ID NO.4, and the corresponding sequence after mutation is ATGACTGCGCCACACTGG. Preferably, the improved soybean seed quality comprises at least one of the following: (1) Regulate the protein content of soybean seeds; (2) Regulate the oil content of soybean seeds; (4) Regulate the egg-to-fat ratio of soybean seeds; (5) Regulate soybean seed size or shape.

9. The method according to claim 8, characterized in that, The method includes the following steps: (1) Design the first sgRNA and the second sgRNA based on the coding sequence of the soybean GmTT8 gene; (2) Construct a GmTT8 dual-target gene editing vector containing the first sgRNA and the second sgRNA; introduce the GmTT8 dual-target gene editing vector into Agrobacterium; (3) Genetic transformation of soybean recipient material using the Agrobacterium; (4) Screening and identification of GmTT8 gene editing positive materials; (5) Identify the GmTT8 gene editing positive materials.

10. The application of the soybean GmTT8 gene as a gene editing target in improving soybean quality, characterized in that... The improved soybean seed quality includes at least one of the following: (1) Regulate the protein content of soybean seeds; (2) Regulate the oil content of soybean seeds; (3) Regulate the egg-to-fat ratio of soybean seeds; (4) Regulate soybean seed size or shape; (5) Regulating the color of the soybean hilum; Preferably, SEQ ID NO.4 and SEQ ID NO.5 in the soybean GmTT8 gene are used as gene editing targets.

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