Use of gm tc1b protein and its encoding gene in improving soybean oil content or / and seed weight

Abstract

This invention belongs to the field of molecular breeding and discloses the application of a GmTC1B protein and its encoding gene in increasing soybean oil content and / or seed weight. This is achieved by inhibiting or deleting the GmTC1B protein. GmTC1B The expression of the gene, or by inhibiting or deleting the activity of the GmTC1B protein, can increase the oil content and / or weight of soybean seeds. A method for breeding soybean varieties with high oil content and / or increased seed weight is also disclosed, comprising inhibiting the expression of the gene in soybeans, or by inhibiting or deleting the activity of the GmTC1B protein. GmTC1B Gene expression or inhibition of GmTC1B protein activity, GmTC1B The nucleotide sequence of the gene is shown in SEQ ID NO:3, and the amino acid sequence of the GmTC1B protein is shown in SEQ ID NO:4. This invention discloses and verifies... GmTC1B The role of the gene or its encoded protein in the regulation of soybean seed oil content and seed weight, by reducing or knocking out the function of the gene, can significantly increase the oil content and 100-seed weight of soybean seeds at the same time. This provides a new key target gene for the synergistic improvement of soybean yield and quality traits through molecular means.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering and molecular breeding, and in particular relates to the application of a GmTC1B protein and its encoding gene in increasing soybean oil content and / or seed weight. Background Technology

[0002] Soybeans Glycine max Soybean is an important oilseed and protein crop. Seed oil content and yield are key agronomic traits that determine its economic value and planting benefits. However, traits such as oil content and yield in soybeans are usually controlled by multiple genes, with a complex genetic basis. Simultaneous improvement of these traits through traditional breeding methods is limited by long cycles and low efficiency.

[0003] Currently, increasing the oil content and yield of major soybean varieties still faces challenges. Identifying key genes that regulate the synthesis and accumulation of oil in soybean seeds and using modern biotechnology to perform targeted genetic improvement is an effective way to create new high-oil, high-yield germplasm and cultivate breakthrough varieties.

[0004] Therefore, identifying key genes that play an important role in regulating soybean oil accumulation and seed size / weight, elucidating their functions, and developing corresponding molecular breeding techniques are of great significance for promoting soybean genetic improvement. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the deficiencies and defects mentioned in the background art above, and to provide an application of GmTC1B protein and its encoding gene in increasing soybean oil content and / or seed weight.

[0006] To solve the above-mentioned technical problems, the technical solution proposed by this invention is as follows: GmTC1B The application of the gene or GmTC1B protein in increasing soybean oil content and / or seed weight, by inhibiting or deleting the gene. GmTC1B The expression of the gene, or by inhibiting or deleting the activity of the GmTC1B protein, can increase the oil content and / or increase the weight of soybean seeds, wherein the amino acid sequence of the GmTC1B protein is as shown in SEQ ID NO: 4; the gene encoding the GmTC1B protein... GmTC1B The nucleotide sequence of the gene is shown in SEQ ID NO: 3.

[0007] As a general inventive concept, the present invention also provides a method for breeding soybean varieties with high oil content and / or increased seed weight, comprising inhibiting... GmTC1B The expression of the gene or the inhibition of the activity of the GmTC1B protein, the aforementioned GmTC1BThe nucleotide sequence of the gene is shown in SEQ ID NO: 3; the amino acid sequence of the GmTC1B protein is shown in SEQ ID NO: 4.

[0008] The above method, preferably, involves the suppression using a CRISPR-Cas9 system. GmTC1B Gene editing is used to achieve this.

[0009] The above method, preferably, uses sgRNA targeting in the CRISPR-Cas9 system. GmTC1B The target sequence of the gene is shown in SEQ ID NO: 5.

[0010] Preferably, the above method includes the following steps: (1) Construction GmTC1B Gene CRISPR / Cas9 knockout vector; (2) The vector obtained in step (1) is introduced into Agrobacterium and then transferred into soybeans for genetic transformation. (3) Identify the soybeans obtained in step (2) to obtain soybeans with high oil content and / or increased seed weight.

[0011] The above method, preferably, the GmTC1B The gene editing vector contains SG1; the SG1 is shown in SEQ ID NO: 5.

[0012] In the above method, preferably, the carrier is pCBSG015.

[0013] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) This invention discloses and verifies GmTC1B The role of the gene or its encoded protein in the regulation of soybean seed oil content and seed weight, by reducing or knocking out the function of the gene, can significantly increase the oil content and 100-seed weight of soybean seeds at the same time. This provides a new key target gene for the synergistic improvement of soybean yield and quality traits through molecular means.

[0014] (2) This invention successfully created a gene editing system using CRISPR / Cas9 gene editing technology. GmTC1B Gene knockout soybean mutants showed a significant increase in seed oil content, as well as a significant increase in seed length, width, thickness, and weight per 100 seeds. This provides a material basis for the subsequent creation of new high-oil and high-yield soybean materials. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0016] Figure 1 A schematic diagram of CRISPR / Cas9 gene editing target sites.

[0017] Figure 2 for GmTC1B A schematic diagram of gene editing vector construction.

[0018] Figure 3 This is a flowchart of soybean genetic transformation.

[0019] Figure 4 T2 generation CRISPR / Cas9 gene-edited plants GmActin Gene PCR detection diagram: M:Maker; 1-6 are gene-edited plants. gmtc1b-1,-2,-3,-4,-5,-6 ;-: negative control.

[0020] Figure 5 T2 generation CRISPR / Cas9 gene-edited plants Cas9 Gene PCR detection diagram: M:Maker; 1-6 are gene-edited plants. gmtc1b-1,-2,-3,-4,-5,-6 ;-: negative control.

[0021] Figure 6 T2 generation CRISPR / Cas9 gene-edited plants GmTC1B Gene PCR detection diagram: M:Maker; 1-6 are gene-edited plants. gmtc1b-1,-2,-3,-4,-5,-6 ;-: negative control.

[0022] Figure 7 Mutation type analysis of T2 generation CRISPR / Cas9 gene-edited plants.

[0023] Figure 8 Oil content of CRISPR / Cas9 gene-edited plants.

[0024] Figure 9 Seed size characteristics of CRISPR / Cas9 gene-edited plants.

[0025] Figure 10 Seed characteristics and 100-seed weight of CRISPR / Cas9 gene-edited plants. Detailed Implementation

[0026] To facilitate understanding of the present invention, the present invention will be described more fully and in detail below with reference to the accompanying drawings and preferred embodiments, but the scope of protection of the present invention is not limited to the following specific embodiments.

[0027] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by those skilled in the art. The technical terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the scope of the invention.

[0028] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be purchased from the market or prepared by existing methods.

[0029] Example: 1. GmTC1B Cloning of genes From the Phytozome public database ( https: / / phytozome-next.jgi.doe.gov / Soybeans obtained from GmTC1B Gene (accession number: Glyma.04G065500 The encoded sequence of ).

[0030] Design and synthesize specific primers for amplifying the gene: Forward primer GmTC1B-F: 5'-ATGAAGCAAGCTTCGTCGGAG-3' (SEQ ID NO: 1) Reverse primer GmTC1B-R: 5'-TCAAGAGTGATTAAACCAGGGTCC-3' (SEQ ID NO: 2) Soybean cDNA was used as a template for PCR amplification using high-fidelity DNA polymerase. The reaction mixture consisted of 25 μL of 2×PhantaMaster Mix, 2.5 μL each of forward and reverse primers, 2 μL of cDNA template, and ddH2O to a total volume of 50 μL. The PCR conditions were as follows: 98℃ pre-denaturation for 5 min; 30 cycles of 98℃ for 30 sec, 60℃ for 30 sec, and 72℃ for 4 min; and a final extension at 72℃ for 5 min.

[0031] DNA was purified and recovered, ligated into a cloning vector, and sequenced. 4 μL of purified DNA and 1 μL of pEASY-Blunt T vector were incubated at 24°C for 25 min. 5 μL of the ligation reaction mixture was added to newly thawed *E. coli* competent cells (Trans1-T1), incubated on ice for 2 min, heat-shocked at 42°C for 1 min, and then incubated on ice again. 1 mL of pre-chilled LB broth (4°C) was added, and the mixture was incubated at 37°C with shaking for 1 h. 200 μL of the bacterial culture was plated onto LB + Kan plates and incubated at 37°C for 16 h. Single colonies were picked for colony PCR. PCR system: 7.5 μL 2×Rapid Taq Master Mix; 5.5 μL ddH2O; forward primers. 35s F: 5'-CAAAGGGTAATATCCGGAAACCTCC-3' (as shown in SEQ ID NO: 11) and reverse primer jc-GmTC1B R: 0.5 μL each of 5'-ATCGTAACCGCCTACCTCGAAGTAC-3' (as shown in SEQ ID NO: 12). PCR amplification conditions were: 98℃ pre-denaturation for 5 min; 30 cycles of 98℃ for 25 sec, 63℃ for 25 sec, and 72℃ for 40 sec; extension at 72℃ for 5 min. Positive single colonies were picked and cultured in 10 mL LB + Kan liquid medium at 37℃ with shaking for 16 h. The bacterial culture samples were then sent for sequencing.

[0032] Obtain the amino acid sequence encoding SEQ ID NO: 4 GmTC1B The gene, whose nucleotide sequence is shown in SEQ ID NO: 3.

[0033] (1) GmTC1B Original nucleotide sequence: (5028bp) SEQ ID NO: 3 ATGGGGCTGTGA TTGCCGGGAGG (2) Original amino acid sequence of GmTC1B: (1676aa) as shown in SEQ ID NO: 4 2. Construction of CRISPR / Cas9 gene editing vectors In the Phytozome public database ( https: / / phytozome-next.jgi.doe.gov / Download from ) GmTC1B ( Glyma.04G065500 The gene sequence was obtained using the website (http: / / crispr.hzau.edu.cn / CRISPR2 / ). GmTC1B Predictive analysis was performed on potential targets of 19nt+PAM (NGG) in the gene.

[0034] Target design: such as Figure 1 As shown, for GmTC1B The first exon region of the gene (SEQ ID NO: 3) was predicted and screened using online tools to obtain a specific sgRNA target, whose target sequence is: ATGGGGCTGTGATTGCCGGGAGG, and the target sequence is denoted as SG1 (as shown in SEQ ID NO: 5).

[0035] Vector construction: A DNA fragment containing the AtU6 promoter-driven sgRNA expression cassette shown in SEQ ID NO: 5 was synthesized (its sequence summary is shown in SEQ ID NO: 6). This fragment was then... Hind III and Sma I The restriction enzyme sites were inserted into the pCBSG015 vector backbone, which had also been double-digested, to construct a recombinant gene editing vector named pCBSG015-. GmTC1B The pCBSG015 carrier skeleton contains CaMV 35S A promoter-driven Cas9 expression unit and a Basta resistance marker for plant screening were used. The vector, pCBSG015 (Basta), was provided by Weimi Biotechnology Co., Ltd., and its resistance in prokaryotes is kanamycin, while its resistance in eukaryotes is glufosinate-ammonium (Basta; PPT). The vector construction diagram is shown below. Figure 2 As shown.

[0036] Gene synthesis contains promoter + GmTC1B One sgRNA tandem expression cassette, sequence as follows: aagctt (HindIII) ttttttgttttacgttgaacaacggaaactcgacttgccttccgcacaatacatcatttcttcttagctttttttcttcttcttcgttcatacagtttttttttgtttatcagcttacattttcttgaaccgtagctttcgttttcttctttttaactttccattcggagtttttgtatcttgtttcatagtttgtcccaggattagaatgattaggcatcgaaccttcaagaatttgattgaataaaacatcttcattcttaagatatgaagataatcttcaaaaggcccctgggaatctgaaagaagagaagcaggcccatttatatgggaaagaacaatagtatttcttatataggcccatttaagttgaaaacaatcttcaaaagtcccacatcgcttagataagaaaacgaagctgagtttatatacagctagagtcgaagtagtgatt (AtU6 promoter) ATGGGGCTGTGATTGCCGGGAGG (Target) gttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgc (sgRNA) ttttttgttttagagcttcggctagtccgtagcgcgtgcgccaattctgcagacaaatggccccggg (SmaI) (shown in SEQ ID NO: 6).

[0037] Agrobacterium transformation: Transfer the pCBSG015- GmTC1B recombinant vector into EHA105 Agrobacterium. Add 2 μL of pCBSG015- GmTC1B recombinant vector to the freshly thawed EHA105Agrobacterium competent cells were incubated on ice for 5 min, flash-frozen in liquid nitrogen for 1 min, heat-shocked in a 37°C water bath for 3 min, and then incubated on ice for 3 min. 1 mL of LB broth was added, and the cells were incubated at 28°C with shaking for 90 min. 200 μL of the bacterial culture was plated onto LB+Kan+Rif+Gen plates and incubated at 28°C for 48 h. Single colonies were picked for colony PCR. The PCR system consisted of: 1 μL template DNA; 7.5 μL 2×Rapid Taq Master Mix; 0.5 μL each of forward and reverse primers (Cas9-detection F and Cas9-detection R); and 5.5 μL ddH2O. The PCR amplification conditions were as follows: 98°C pre-denaturation for 5 min; 30 cycles of 98°C for 30 sec, 63°C for 30 sec, and 72°C for 30 sec; and extension at 72°C for 5 min. A positive Agrobacterium strain was obtained for soybean genetic transformation.

[0038] Forward primer Cas9-detection F: 5'-CCGACGAGTACAAGGTGCCCA-3' (as shown in SEQ ID NO: 13); Reverse primer Cas9-detection R: 5'-GGCGATCAGATTTTCCAGCC-3' (as shown in SEQ ID NO: 14).

[0039] 3. Soybean genetic transformation and acquisition of transgenic plants Soybean genetic transformation flowchart as follows Figure 3 As shown.

[0040] Explant preparation and infection: Seeds of the soybean cultivar Tianlong No. 1 were taken and surface-sterilized (the seeds were placed in a sterile 100mL Erlenmeyer flask and rinsed twice with 75% alcohol for sterilization. Then, 25mL of 84 disinfectant, 75mL of sterile water, and 3 drops of Tween-20 were added, and the seeds were soaked for 15 minutes). After rinsing three times with sterile water, the seeds were placed hilum-side down on germination medium (GM) and germinated for 2 days under light (26℃, 18h light / 6h dark light cycle). The germination medium (GM) consisted of 1 / 2MS salt ions + 20g / L sucrose + 7g / L agar, pH 5.8, and was autoclaved at 120℃. The seed coat was peeled off, and the hypocotyl end was removed to prepare semi-seed explants.

[0041] Preparation of Agrobacterium culture: Select a single positive colony and incubate in 2 mL of YEP medium containing antibiotics with shaking for 12 h (250 rpm, 28℃). Add 0.2 mL of saturated culture to 250 mL of YEP medium containing antibiotics and incubate overnight until the logarithmic growth phase (OD200). 650=0.3~0.6). Collect the bacterial pellet by centrifugation, and resuspend the pellet in liquid co-culture medium (CM) to OD. 650 The pH was 0.6. YEP medium: 5 g / L NaCl + 5 g / L yeast extract + 10 g / L peptone + 15 g / L agar, autoclaved. Co-culture medium (CM): 1 / 2 MS salt ions + 3.9 g / L MES + 30 g / L sucrose + vitamin B5 + 153 mg / L DTT + 2 mg / L zeatin + 40 mg / L AS + 7 g / L agar, pH 5.4, autoclaved.

[0042] Agrobacterium co-culture: Place 30 ml of Agrobacterium into a culture dish, and prepare 50 explants for each Agrobacterium culture dish, ensuring that the explants are completely suspended in the bacterial solution. After 30 min of incubation, use sterile forceps to transfer the explants to a solid co-culture medium, 15 explants per dish, placed horizontally, sealed with breathable tape, and placed in an incubator (23℃) for dark incubation for 5 days.

[0043] Shoot induction and selection: After co-culture, explants were first transferred to stem induction medium SI (15 explants per dish). The dishes were sealed with breathable tape and cultured under light for 7 days (26℃, 18h light / 6h dark photocycle). Then, the elongated cotyledonary hypocotyls were removed, and the explant tissues were transferred to SI medium containing selection agents and cultured under light for 21 days (7 explants per dish). Stem induction medium (SI): B5 salt ions + 0.98 g / L MES + 30 g / L sucrose + B5 vitamin + 150 mg / L cephalosporin + 450 mg / L temetin + 1 mg / L 6-BA + 50 mg / L asparagine + 50 mg / L glutamine + 6 mg / L glufosinate + 7 g / L agar, pH 5.7, autoclaved.

[0044] Stem elongation: Transfer differentiated explants to stem elongation (SE) medium. Remove the cotyledons of the explants and make a fresh incision at the base of the developing node. Transfer the explants to fresh stem elongation (SE) medium and culture at 26°C for 6 weeks under light. Change the SE medium every 2 weeks. Make a fresh horizontal incision at the base of the explant each time the medium is changed. Stem elongation (SE) medium: MS salts + 0.6 g / L MES + 30 g / L sucrose + vitamin B5 + 150 mg / L cephalosporin + 450 mg / L temetin + 0.1 mg / L IAA + 0.5 mg / L GA + 1 mg / L zeatin + 50 mg / L asparagine + 50 mg / L glutamine + 6 mg / L glufosinate + 7 g / L agar, pH 5.7, autoclaved.

[0045] Rooting: When the stems reach 3 cm in length, cut them from the tissue, soak them in IBA (1 mg / ml) for 2 minutes, and then transfer them to glass bottles containing rooting medium (RM) for further cultivation. After 2 weeks, when more than 2 roots have grown from the stems, transplant them into the soil and harden them off. Cultivate at 26℃ under light for about 1 week until the seedlings survive. Rooting medium (RM): 1 / 2 MS salt ions + 0.6 g / L LMES + 20 g / L sucrose + vitamin B5 + 3 mg / L glufosinate + 7 g / L agar, pH 5.7, autoclaved.

[0046] Hardening off and transplanting: After the root system has developed well, transplant the complete plant into nutrient soil and cultivate it in a greenhouse until it matures and bears fruit, and harvest the T0 generation seeds.

[0047] 4. Screening and gene editing identification of transgenic plants PCR identification of transgenic positive plants: Genomic DNA was extracted from T0, T1, and T2 generation resistant plants. PCR detection was performed using forward primer Cas9-detection F and reverse primer Cas9-detection R (PCR amplification reaction conditions were as follows: 98℃ pre-denaturation for 5 min; 98℃ for 30 sec, 63℃ for 30 sec, 72℃ for 30 sec, 30 cycles; 72℃ extension for 5 min). Plants that amplified the expected band size were considered transgenic plants with inserted T-DNA fragments, while those without the target product were considered transgenic plants without inserted T-DNA fragments. Electrophoresis results are shown below. Figure 5 As shown.

[0048] Editing type identification: Total DNA was extracted from positive plants, and the quality of the DNA was detected by PCR using primers GmActin F: 5'-CGGTGGTTCTATCTTGGCATC-3' (as shown in SEQ ID NO: 15) / GmActin R: 5'-GTCTTTCGCTTCAATAACCCTA-3' (as shown in SEQ ID NO: 16). The reaction system was as follows: template DNA 1 μL; 2×Rapid Taq Master Mix 7.5 μL; forward and reverse primers 0.5 μL each; ddH2O 5.5 μL. The preferred PCR reaction conditions were: 98℃ pre-denaturation for 5 min; 30 cycles of 98℃ for 30 sec, 56℃ for 30 sec, and 72℃ for 30 sec; extension at 72℃ for 5 min. The electrophoresis image of the PCR product is shown below. Figure 4 As shown.

[0049] use GmTC1BPrimers TC1b-F (5'-CCAAATTGGGTTACCTCTTCAACAC-3', located 160 bp upstream of the gene target site, as shown in SEQ ID NO: 17) / TC1b-R (5'-ATAAGTCGACAATCCCGATTACCAA-3', as shown in SEQ ID NO: 18) were used to amplify the DNA. The amplified PCR products were then sequenced for analysis. The reaction system was as follows: template DNA 1 μL, 2×Rapid Taq Master Mix 7.5 μL, forward and reverse primers 0.5 μL each, ddH2O 5.5 μL; the preferred reaction conditions were: 98℃ pre-denaturation for 5 min; 30 cycles of 98℃ for 30 sec, 58℃ for 30 sec, 72℃ for 30 sec; extension at 72℃ for 5 min. The electrophoresis results of the PCR products are shown below. Figure 6 As shown.

[0050] The sequencing results were compared with the wild-type sequence (SEQ ID NO: 3) to identify the mutation type.

[0051] The specific results of the sequencing and alignment analysis are as follows: Figure 7 As shown, two strains without exogenous origin were identified. Cas9 T2 generation CRISPR / Cas9 gene-edited plant material, numbered as follows gmtc1b-1 , gmtc1b-6 Inside the plant GmTC1B All genes underwent effective editing and were identified as homozygous mutations. Among them, in gmtc1b-1 In the plant, GmTC1B A 10-base deletion (-10 bp deletion) in the gene leads to a frameshift mutation, premature termination of translation, and the amino acid sequence of the encoded protein GmTC1B-1 becomes 408aa; gmtc1b-6 In the plant, GmTC1B The deletion of one base (-1 bp deletion) leads to a frameshift mutation in the gene's reading frame, causing premature termination of translation and affecting the protein it encodes. GmTC1B-2 The amino acid sequence changed to 411aa. Both mutations resulted in reading frame shifts, causing... GmTC1B Protein translation terminates prematurely (the mutant proteins are 408 and 411 amino acids long, respectively), thus completely knocking out the function of the gene.

[0052] wild type GmTC1B The two different mutant alleles produced after gene editing are respectively GmTC1B-1 and GmTC1B-2 , GmTC1B-1 The nucleotide sequence is shown in SEQ ID NO: 7, and the amino acid sequence is shown in SEQ ID NO: 8. GmTC1B-2The nucleotide sequence is shown in SEQ ID NO: 9, and the amino acid sequence is shown in SEQ ID NO: 10. The sequences SEQ ID NO: 7 to 10 were derived using bioinformatics methods based on SEQ ID NO: 1 and the aforementioned local mutation site information.

[0053] (1) GmTC1B-1 Nucleotide sequence: (5018bp) SEQ ID NO: 7 ATGGGGCGGGAG G (2) GmTC1B-1 Amino acid sequence: (408aa) SEQ ID NO: 8 MKQASSEAVPSLSSAPSFTEAATSSTSSAAAAEDLAVGSRDGGGGAQETVAVDRRGEYSAVCRWTVHNFPRIKARALWSKYFEVGGYDCRLLIYPKGDSQALPGYISIYLQIMDPRGTSSSKWDCFASYRLAIVNLADDSKTIHRDSWHRFSSKKKSHGWCDFTPSNTVFDPKLGYLFNTDSVLITADILILNESVNFTRDNNEVQSSSSSSSSAMTSSVVASPVSDVSSGKFTWKVHNFSLFKEMIKTQKIMSPVFPAGECNLRISVYQSSVNGVEYLSMCLESKDTDKSVVLSDRSCWCLFRMSVLNQKPGSNHMHRDSYGRFAADNKSGDNTSLGWNDYMKMLDFIDADSGFLVDDTAVFSTSFHVIKEFSSFSKNGAGGVGVVQGSLMDMLESSLGGLRILLG* (3) GmTC1B-2 Nucleotide sequence: (5027bp) SEQ ID NO: 9 ATGGGGCTGTGA TTGCGGGAGG (4) GmTC1B-2 Amino acid sequence: (411aa) SEQ ID NO: 10 MKQASSEAVPSLSSAPSFTEAATSSTSSAAAAEDLAVGSRDGGGGAQETVAVDRRGEYSAVCRWTVHNFPRIKARALWSKYFEVGGYDCRLLIYPKGDSQALPGYISIYLQIMDPRGTSSSKWDCFASYRLAIVNLADDSKTIHRDSWHRFSSKKKSHGWCDFTPSNTVFDPKLGYLFNTDSVLITADILILNESVNFTRDNNEV QSSSSSSSSAMTSSVVASPVSDVSSGKFTWKVHNFSLFKEMIKTQKIMSPVFPAGECNLRISVYQSSVNGVEYLSMCLESKDTDKSVVLSDRSCWCLFRMSVL NQKPGSNHMHRDSYGRFAADNKSGDNTSLGWNDYMKMLDFIDADSGFLVDDTAVFSTSFHVIKEFSSFSKNGAVIAGGVGVVQGSLMDMLESSLGGLRILLG* 5. Phenotypic analysis of gene-edited mutants Seed oil content determination: Near-infrared cereal analyzer was used to analyze the wild-type (WT) and T2 generation homozygous mutants. gmtc1b-1 , gmtc1b-6 The oil content of mature seeds was determined. The results are as follows: Figure 8 As shown, the average oil content of wild-type soybean seeds is 19.98%, while... gmtc1b-1 and gmtc1b-6 The oil content of the mutant seeds was significantly increased to 23.29% and 22.33%, respectively.

[0054] Seed morphology and 100-seed weight analysis: Ten seeds were randomly selected from harvested T2 generation CRISPR / Cas9 gene-edited soybean seeds for statistical analysis of length, width, and thickness. Results showed that the mutant... gmtc1b-1 and gmtc1b-6 The length, width, and thickness of each grain are significantly greater than WT. Figure 9 Therefore, soybean seeds become larger after the GmTC1B protein is missing.

[0055] One hundred soybean seeds were randomly selected from harvested CRISPR / Cas9 gene-edited soybean seeds for statistical analysis of their 100-seed weight. The results showed that the total weight (WT) of 100 soybean seeds in the control group was approximately 18.1 g. gmtc1b-1 One hundred grains weigh approximately 21.3g. gmtc1b-6 The weight of 100 grains is approximately 21.8g. Figure 10 The 100-seed weight of CRISPR / Cas9 gene-edited soybeans was significantly greater than that of the control group. Therefore, the deletion of GmTC1B protein increased the 100-seed weight of soybeans.

[0056] Conclusion: The above examples confirm that CRISPR-Cas9 technology can be used to knock out soybeans. GmTC1B Genes can be used to obtain homozygous mutants that are stably inherited. These mutants... GmTC1B In the case of complete loss of gene function, it exhibits two excellent agronomic traits: significantly increased seed oil content, increased seed volume, and significantly increased 100-seed weight.

Claims

1. GmTC1B The application of the gene or GmTC1B protein in increasing soybean oil content and / or seed weight, characterized in that... By suppressing or deleting the above GmTC1B The expression of the gene, or by inhibiting or deleting the activity of the GmTC1B protein, can increase the oil content and / or increase the weight of soybean seeds, wherein the amino acid sequence of the GmTC1B protein is as shown in SEQ ID NO: 4; the encoding of the GmTC1B protein is... GmTC1B The nucleotide sequence of the gene is shown in SEQ ID NO:

3.

2. A method for breeding soybean varieties with high oil content and / or increased seed weight, characterized in that, Including inhibition in soybeans GmTC1B The expression of the gene or the inhibition of the activity of the GmTC1B protein, the aforementioned GmTC1B The nucleotide sequence of the gene is shown in SEQ ID NO: 3; the amino acid sequence of the GmTC1B protein is shown in SEQ ID NO:

4.

3. The method as described in claim 2, characterized in that, The inhibition was achieved by using the CRISPR-Cas9 system. GmTC1B Gene editing is used to achieve this.

4. The method as described in claim 3, characterized in that, The CRISPR-Cas9 system uses sgRNA targeting GmTC1B The target sequence of the gene is shown in SEQ ID NO:

5.

5. The method as described in claim 2, characterized in that, The method includes the following steps: (1) Construction GmTC1B Gene CRISPR / Cas9 knockout vector; (2) The vector obtained in step (1) is introduced into Agrobacterium and then transferred into soybeans for genetic transformation. (3) Identify the soybeans obtained in step (2) to obtain soybeans with high oil content and / or increased seed weight.

6. The method as described in claim 2, characterized in that, The GmTC1B The gene editing vector contains SG1; the SG1 is shown in SEQ ID NO:

5.

7. The method according to claim 6, characterized in that, The carrier is pCBSG015.

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