Application of B3 transcription factor TaFUS3 in increasing starch content of wheat grains

Abstract

The invention belongs to the technical field of plant genetic engineering, and particularly relates to application of a B3 transcription factor TaFUS3 in regulating and controlling the starch content of wheat grains. According to the invention, a wheat TaFUS3 gene mutant strain is created by using a CRISPR / Cas9 technology, and an overexpression strain is obtained through transgenosis. The TaFUS3 gene overexpression, the TaFUS3 expression level of a mutant material and the total starch content, the amylose content and the amylopectin content of the wheat grains are determined, and it is found that after the TaFUS3 gene overexpression, the total starch content, the amylose content and the amylopectin content of the wheat grains are obviously increased. The result shows that the TaFUS3 gene promotes the accumulation of wheat grain starch and is beneficial to the metabolic synthesis of carbon.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, specifically relating to the application of B3 transcription factor TaFUS3 in regulating wheat grain starch content. Background Technology

[0002] Wheat is widely cultivated and provides humans with essential carbohydrates, proteins, and nutrients. In addition to its crucial role in food processing, wheat also plays a vital role in animal feed, industrial raw materials, and biofuels, making it essential for food security and economic development.

[0003] Starch is the main component of wheat grains and can be divided into amylose and amylopectin based on their molecular structure. FUSCA3 (FUS3) is a B3 transcription factor that plays an important role in seed development, maturation, after-ripening, and germination. Currently, there are no reports on the regulation of wheat grain starch content by the B3 transcription factor TaFUS3. Summary of the Invention

[0004] This invention proposes the application of B3 transcription factor TaFUS3 in regulating wheat grain starch content.

[0005] This invention discovers that overexpression in wheat receptors TaFUS3 After gene modification, the total starch, amylose, and amylopectin content of wheat grains was significantly increased. Furthermore, the use of CRISPR / Cas9 technology to modify wheat... TaFUS3 After gene editing, it was found that... TaFUS3 Gene editing did not affect the tissues examined. TaFUS3 Gene transcription.

[0006] Therefore, the first aspect of this invention provides the application of the B3 transcription factor TaFUS3, a nucleic acid encoding TaFUS3, and biological materials containing said nucleic acid in regulating the starch content of wheat grains. The biological material is an expression cassette, transposon, plasmid vector, viral vector, or host cell. The amino acid sequence of TaFUS3 is shown in SEQ ID NO:1, and the sequence of the nucleic acid encoding TaFUS3 is shown in SEQ ID NO:2.

[0007] The present invention also provides the application of B3 transcription factor TaFUS3, nucleic acid encoding TaFUS3, and biological material containing said nucleic acid in the cultivation of wheat with increased grain starch content.

[0008] This invention also provides a method for increasing the starch content in wheat grains, comprising: By increasing the content and / or activity of the B3 transcription factor TaFUS3 in the recipient wheat, overexpressed wheat is obtained, wherein the starch content of the grains of the overexpressed wheat is higher than that of the recipient wheat.

[0009] One method to increase the content and / or activity of the B3 transcription factor TaFUS3 in recipient wheat is to overexpress the TaFUS3 protein in recipient wheat; the overexpression method involves introducing a nucleic acid molecule encoding the TaFUS3 protein into recipient wheat. Specifically, Agrobacterium-mediated transformation can be used to introduce the nucleic acid molecule encoding the TaFUS3 protein into recipient wheat.

[0010] The beneficial effects of this invention are as follows: This invention discovers a novel function of the B3 transcription factor TaFUS3: its ability to regulate starch content in wheat grains. Overexpression in the wheat receptor... TaFUS3 Following gene administration, the total starch, amylose, and amylopectin content of wheat grains was significantly increased. This indicates that TaFUS3 promotes starch accumulation in wheat grains, which is beneficial for carbon metabolism and synthesis. Attached Figure Description

[0011] Picture 1 : Map of the overexpression backbone vector pLGY-02.

[0012] Picture 2 Map of the intermediate vector pCBC-MT1T2 used for gene editing.

[0013] Picture 3 Map of the pLGYE-002 backbone vector used for gene editing.

[0014] Picture 4 CRISPR / Cas9-FUS3 vector map.

[0015] Picture 5 Determination of linear and amylopectin using dual wavelengths.

[0016] Picture 6 Wheat overexpression TaFUS3 Identification of selection marker genes in plants (T6 generation). M: molecular weight standard DL2000; 1-21: transgenic plants; 22: blank control; 23: negative control; 24: positive control.

[0017] Picture 7 Overexpression TaFUS3 Identification of the target gene in wheat plants (T6 generation). M: molecular weight standard DL2000; 1-21: transgenic plants; 22: blank control; 23: negative control; 24: positive control.

[0018] Picture 8 : TaFUS3 Identification of gene-edited plants (T4 generation). M: Molecular weight standard DL2000; 1-19: fus3 Gene-edited plants; 20: blank control.

[0019] Picture 9 Endogenous factors during grain development TaFUS3 Expression characteristics in overexpression and gene-edited lines.

[0020] Picture 10 During the germination process of seeds TaFUS3 Expression characteristics in overexpression and gene-edited lines. Detailed Implementation

[0021] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions in the embodiments of this invention will be clearly described below. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0022] The wild wheat 'JW1' used in the examples was provided by the Shandong Academy of Agricultural Sciences.

[0023] Some of the experimental reagents used in the examples: TB Green® Premix Ex Taq™ II (Tli RNaseH Plus), PrimeScript™ RTreagent Kit with gDNA Eraser, DL2000 Marker, DL5000 Marker, etc. were all purchased from TaKaRa; TransZol Plant was purchased from Beijing TransGen Biotech Co., Ltd.; 2*Es Taq MasterMix (Dye) was purchased from Kangwei Century Co., Ltd.; amylose standards and amylopectin standards were purchased from Solarbio; agarose was purchased from Sangon Biotech Co., Ltd.; PCR primer synthesis was performed by Shangya Biotechnology Co., Ltd.

[0024] Example 1 TaFUS3 Overexpression and CRISPR / Cas9 gene editing TaFUS3 The wheat material was obtained by genetically transforming wheat embryos using JW1 as the acceptor through Agrobacterium tumefaciens-mediated transformation, and was prepared by the Shandong Academy of Agricultural Sciences. The public can also commission the Shandong Academy of Agricultural Sciences to prepare wheat material JW1 as the acceptor. TaFUS3 overexpression and TaFUS3 Gene-edited wheat.

[0025] 1. TaFUS3 Preparation of wheat overexpression materials Specifically, the following steps are included: (1) Construction of overexpression vector The overexpression vector backbone used was the pLGY-02 vector ( Picture 1 The pLGY-02 vector is placed between the restriction enzyme sites. Kpn I and Spe The components between I are replaced with TaFUS3 Partially, the overexpression vector pLGY-FUS3 was obtained. The overexpression vector pLGY-FUS3 contains... Hyg Marker genes.

[0026] (2) Transformation of Agrobacterium The constructed gene overexpression vector pLGY-FUS3 was transformed into competent cells of Agrobacterium strain EHA105. The bacterial culture was spread on LB solid medium (containing 50 mg / L Kan) under a clean bench and incubated at 28 ℃. The growth was detected after 2-3 days.

[0027] (3) Infecting wheat callus tissue and promoting regeneration The successfully transformed Agrobacterium was cultured at 28°C until OD600≈0.5-0.8. During this period, bacterial culture was identified by PCR using primers F2: 5'-GGTACCATGGCCGCCATCA-3' (SEQ ID NO:3) and R2: 5'-ACTAGTTCACATCAGAGGCC-3' (SEQ ID NO:4). The correctly identified positive bacterial culture was then used to infect wheat callus tissue. Transgenic plants were obtained through embryogenic callus induction and plant regeneration.

[0028] (4) Identification of T0 generation plants overexpressing T0 PCR identification was performed using specific primers OVER-FUS3-F1 (5'-CGACCAGTAAATGAGCTGGATT-3', SEQ ID NO:5) / OVER-FUS3-R1 (5'-CAGACTTTGGGTTGGGAAACA-3', SEQ ID NO:6) and hygF1 (5'-CGAGTACTTCTACACAGCCATC-3', SEQ ID NO:7) / hygR1 (5'-GTCTGTCGAGAAGTTTCTGATCG-3', SEQ ID NO:8) (primer information is shown in Table 1). A total of 18 T0 generation positive plants were obtained, designated as OE-1, 2…18.

[0029] 2. TaFUS3 Preparation of gene-edited wheat materials Includes the following steps: (1) Target site design Based on the scoring and specificity, TaFUS3Two suitable target sites were selected in the conserved regions of the second and third exons of the A, B, and D sequences of the gene. Suitable sequence fragments were selected before the PAM structure and set as target sequences as follows: sgRNA1: 5'-GTTGCGAGTTATTCTGCAGA-3' (SEQ ID NO:9); sgRNA2: 5'-CCTCCCAATTCTGACATCAA-3' (SEQ ID NO:10).

[0030] (2) Construction of CRISPR / Cas9-TaFUS3 gene editing vector First, PCR amplification was performed using the intermediate vector pCBC-MT1T2 as a template. The amplification... TaFUS3 The primer for sgRNA1 of the gene corresponds to MT1T2-F1 (5'- AATAATGGTCTCAGGCG GTTGCGAGTTATTCTGCAGA-3', SEQ IDNO:11) and MT1T2-F0 (5'-GGTTGCGAGTTATTCTGAGA GTTTTAGAGCTGAAATAGC -3', SEQ ID NO:12). The primers for amplifying sgRNA2 are MT1T2-R0 (5'-TTGATGTCAGAATTGGGAGG). CGCTTCTTGGTGCC -3', SEQ ID NO:13) and MT1T2-R1 (5'- ATTATTGGTCT TAAAC TTGATGTCAGAATTGGGAGG-3' (SEQ ID NO:14). It should be noted that the underlined bases in the primers are sequences that match the vector pCBC-MT1T2. Bsa I restriction enzyme site. Then, the PCR fragment was purified and recovered. The recovered PCR fragment and the backbone vector pLGYE-002 were then processed with restriction endonucleases. BsaI. Restriction digestion. The linearized pLGYE-002 vector plasmid and the digested PCR product were recovered by electrophoresis. The recovered PCR fragments and the linearized pLGYE-002 vector plasmid were ligated overnight at 37°C using T4 ligase. The ligation product was transformed into *E. coli* DH5α, and positive clones were screened on LB agar plates containing chloramphenicol. Single colonies were picked and cultured in LB liquid medium for 4-6 hours. Colony PCR was performed using pLGYE-FUS3-F1 (5'-ggagtgagtacggtgtgcAGAAAAGGAAACAGTGTTAATC-3', SEQ ID NO:15) and pLGYE-FUS3-R1 (5'-gagttggatgctggatggGAAAAACAATTTGTCCAACC-3', SEQ ID NO:16) for verification. Plasmid extraction was then performed for restriction enzyme digestion verification. Positive clones with correct sequencing were sent to the company for further sequencing verification. Correct sequencing indicates successful construction of the CRISPR / Cas9-FUS3 vector.

[0031] (3) Transformation of Agrobacterium The constructed gene-editing vector CRISPR / Cas9-FUS3 was transformed into competent cells of Agrobacterium strain EHA105. The bacterial culture was spread on LB solid medium (containing 50 mg / L Kan) under a clean bench and cultured in a constant temperature incubator at 28℃. The growth was detected after 2-3 days.

[0032] (4) Infecting wheat callus tissue and promoting regeneration The successfully transformed Agrobacterium was incubated at 28°C until OD200 was reached. 600 The amount of protein is approximately 0.5-0.8, which is then used to infect wheat callus tissue. Transgenic plants are obtained through embryogenic callus induction and plant regeneration.

[0033] (5) Identification of gene-edited T0 generation plants Specific primers pLGYE-FUS3-F1 and pLGYE-FUS3-R1 were used to amplify the target site sequence using DNA from transgenic single plant leaves as a template. Two rounds of PCR amplification were performed according to the Hi-Tom kit instructions. Sequencing and alignment with wild-type sequences confirmed the mutant genotype. A total of 22 T0 generation mutant plants were obtained, denoted as […]. fus3- 1, 2, 3...22.

[0034] The intermediate vector pCBC-MT1T2 used for gene editing ( Picture 2 ) and skeleton carrier pLGYE-002 ( Picture 3 All data were provided by Li Genying of the Shandong Academy of Agricultural Sciences. The CRISPR / Cas9-FUS3 vector is as follows: Picture 4 As shown.

[0035] Example 2 1. Plant materials T0 generation transgenic plants were planted to obtain T1 generation plants. The T1 generation plants were then identified by cutting a 2-3 cm section of a leaf below the heart leaf when the wheat reached the two-leaf stage. The leaf was retrieved in an ice box, flash-frozen in liquid nitrogen, and stored at -80°C for identification of transgenic materials. This process of planting and screening was repeated for each generation until T6 generation transgenic plants were obtained.

[0036] The transgenic materials used in this embodiment are: T4, T5, and T6 generations obtained using 'JW1' as the recipient. TaFUS3 The overexpression of genes in wheat lines OE-4, OE-8, and OE-16, as well as the T4, T5, and T6 gene-edited lines obtained using 'JW1' as the recipient. fus3 -8、 fus3 -9、 fus3 -12、 fus3 -14.

[0037] The experimental materials were planted in the Science and Education Park of Henan Agricultural University using pot cultivation to ensure a consistent growth environment. A randomized block design was employed, with 20 pots of each material planted in three plots, each plot containing 4*10 pots. The pots were 30 cm in diameter and 35 cm deep, each containing 15 kg of dry, sandy loam soil. The soil for the pots was taken from the 0-30 cm topsoil layer of the field and sieved before filling. Before filling the pots, pesticides and fertilizers for soil pests were mixed in, with each pot receiving 50% + 50% (for jointing and heading stages) pure N 2.92 g, K2O 3.1 g, and P2O5 4.04 g. The pots were then planted in the field. Sowing took place around November 4th, 2021, 2022, and 2023, with seedlings thinned to 10 plants per pot at the three-leaf stage (two leaves and one bud). Management remained consistent throughout the entire growth period.

[0038] Wheat ears that flowered on the same day were tagged and labeled. Wheat ears were harvested at 15, 20, 25, 30 and 35 days after flowering, about 20 cm below the stem to maintain ear viability. The collected samples were preserved in an ice box, and the seeds of the first and second florets of the 4-10 spikelets in the middle were immediately extracted, flash-frozen in liquid nitrogen, and then stored at -80℃. RNA was extracted for gene expression analysis during grain development.

[0039] During the physiological maturity stage of wheat, the seeds of the first and second florets of the 4th to 10th spikelets in the middle of the ear were promptly harvested. The seeds were first disinfected with 75% alcohol for 2 minutes, rinsed three times with distilled water, then disinfected with 0.1% HgCl2 for 10 minutes, rinsed 5-6 times with sterile water, and finally placed in disposable petri dishes lined with two layers of sterile filter paper. An appropriate amount of distilled water was added, and the dishes were placed in an incubator in the dark at 25°C. Samples were taken at 0, 12, 24, and 48 hours, flash-frozen in liquid nitrogen, and stored at -80°C for analysis of expression characteristics during seed germination. Each experiment was replicated three times.

[0040] Wheat was harvested promptly after it was fully mature, and the seeds were stored at room temperature for one month before being used to determine the starch and its component content.

[0041] 2. Test Methods 2.1 Extraction of genomic DNA DNA was extracted from wheat leaves using the CTAB method.

[0042] 2.2 Identification of transgenic materials Using DNA from transgenic and wild-type wheat as templates, PCR was used to identify overexpression and gene-edited progeny materials. The PCR primers are shown in Table 1. Statistical analysis was performed using Excel 2021, and chi-square tests and significance analyses were conducted using IBM SPSS 23.

[0043] Table 1 Primers used for the identification of transgenic materials 2.3 Extraction of total RNA RNA was extracted from wheat grains using the TransZol Plant kit from TRANSGEN.

[0044] 2.4 cDNA First-Strand Synthesis Using the extracted RNA as a template, cDNA first-strand synthesis was performed in accordance with the instructions for the TaKaRa PrimeScript™ RT reagent Kit with gDNA Eraser.

[0045] 2.5 Real-time quantitative RT-PCR (qRT-PCR) Quantitative RT-PCR was performed according to the instructions for the TaKaRa kit TB Green® Premix Ex Taq™ II (TliRNaseH Plus). Primers used for the target gene expression characterization analysis are shown in Table 2.

[0046] Table 2 Primers used for target gene expression characterization analysis 2.6 Determination of starch and its components content The content of starch and its components was determined using a dual-wavelength method.

[0047] (1) Reagent preparation: Iodine reagent: Weigh 2.0000 g of potassium iodide, dissolve it in double-distilled water to form a saturated solution, then add 0.2000 g of iodine. After it is completely dissolved, transfer the solution to a 100 mL volumetric flask, make up to volume, store in the dark, and prepare fresh before use.

[0048] Preparation of amylose standard solution: Weigh 0.1000 g of amylose into a 100 mL beaker, add 10 mL of 0.5 mol / L KOH, place in a water bath at 60 °C and react for 10 min, stirring constantly to ensure full reaction. After the reaction is complete, transfer to a 50 mL volumetric flask, add distilled water to make up to volume, shake to mix, and let stand. Prepare the standard curve according to Table 3.

[0049] Preparation of amylopectin standard solution: Weigh 0.1000 g of amylopectin into a 100 mL beaker, add 10 mL of 0.5 mol / L KOH, place in a water bath at 60℃ and react for 10 min, stirring constantly to ensure full reaction. After the reaction is complete, transfer to a 50 mL volumetric flask, add distilled water to make up to volume, shake to mix, and let stand. Prepare the standard curve according to Table 4.

[0050] Table 3. Preparation of the standard curve for amylose Table 4 Preparation of the standard curve for amylopectin Preparation of starch scanning solution: Take 1.3 mL and 2.0 mL of amylose and amylopectin standard solutions respectively and put them into 100 mL beakers. Add 25 mL of distilled water and adjust the pH to 3.5 with 0.1 mol / L HCl solution. Pour the solution into a 50 mL volumetric flask, add 0.5 mL of iodine reagent, and dilute to volume with distilled water. Let stand at 20℃ for 30 min.

[0051] (2) Sample processing: After the mature grains are harvested, they are placed at room temperature for one month. The mature grains are then ground into flour using a small milling machine and passed through a 60-mesh sieve to remove the bran from the flour.

[0052] (3) Extraction of the sample solution: Accurately weigh 0.100 g of flour using a 0.1 g balance and place it in a centrifuge tube. Add an appropriate amount of ether to defatt the flour, then add 10 mL of 0.5 mol / L KOH to the centrifuge tube and place it in a water bath at 60°C for 10 min, stirring constantly to ensure a complete reaction. After the reaction, add distilled water to a 50 mL volumetric flask, shake to mix, and let stand. Take 2.5 mL of the supernatant and place it in a 100 mL beaker. Add 25 mL of distilled water and adjust the pH to 3.5 with 0.1 mol / L HCl solution. Pour the solution into a 50 mL volumetric flask, add 0.5 mL of iodine reagent, and dilute to volume with distilled water. Let stand at 20°C for 30 min.

[0053] (4) Selection of wavelengths and reference wavelengths for the determination of amylose and amylopectin: Spectroscopic scanning was used to measure the starch solution. A graphical method was used to select the dual wavelengths for the determination of amylose and amylopectin: 627 nm and 453 nm for amylose, and 750 nm and 542 nm for amylopectin. Picture 5 ).

[0054] (5) Use a spectrophotometer to measure the absorbance of the sample at wavelengths of 750 nm, 542 nm, 627 nm and 453 nm.

[0055] (6) Calculation of results: In the formula: W is the total starch content; W1 is the amylose content; W2 is the amylopectin content; M is the sample mass; X is the wheat moisture content; and 10 is the unit conversion factor.

[0056] 3. TaFUS3 Identification of transgenic progeny materials and their effects on grain starch and its component content 3.1 Wheat TaFUS3 Identification of overexpression and gene-edited offspring 3.1.1 Wheat TaFUS3 Identification of overexpression progeny materials To clarify whether the materials used in the experiment were genuine transgenic materials, T4, T5, and T6 generations were used. TaFUS3 Using wheat lines OE-4, OE-8, and OE-16 as materials, DNA was extracted from leaves when wheat seedlings reached the two-leaf-one-heart stage. Specific primers hygF1 / hygR1 and OVER-FUS3-F1 / OVER-FUS3-R1 (primer information is shown in Table 1) were used to screen for marker genes. Hyg PCR amplification of the target gene and other genes were performed; some amplification results are shown below. Picture 6 , Picture 7 .from Picture 6 and Picture 7As can be seen, specific bands consistent with the plasmid DNA amplification products were observed by electrophoresis at both the 1 kb and 750 bp molecular weights. Statistical analysis of the identification results (Table 5) shows that... TaFUS3 The foreign gene introduced into the overexpression wheat line can be stably inherited to the offspring. Chi-square test showed that the number of transgenic positive and negative plants in the T4 and T5 generations conformed to Mendelian single gene segregation law of 3:1. After multiple generations of screening, OE-4, OE-8 and OE-16 in the T6 generation have become homozygous, providing reliable materials for subsequent research.

[0057] Table 5 (Transfer) TaFUS3 Identification of overexpressing plants and chi-square test Note: In Table 5 P 0.05 =3.84.

[0058] 3.1.2 Wheat TaFUS3 Identification of gene-edited progeny materials Four gene-edited lines with mutations from generation T4 were selected and planted. When the plants reached the two-leaf-one-heart stage, genomic DNA extracted from wheat leaves was used as a template for PCR amplification using specific primers pLGYE-FUS3-F1 / pLGYE-FUS3-R1 (primer information is shown in Table 1). Partial amplification results are shown below. Picture 8 .from Picture 8 As can be seen, there is a band in the molecular weight range of 250-500 bp that matches the known sequence size. The remaining amplified products were subjected to high-throughput sequencing, and the sequencing results were compared with those of wild-type wheat using the software DNAMAN. TaFUS3 Gene sequences were compared to determine whether the transgenic wheat had mutations, the type of mutation, and the chromosome where the mutation occurred. The sequence comparison results are shown in Table 6. Table 6 shows that all lines showed mutations of varying degrees. No mutations were found in sgRNA1. All mutations in sgRNA2 were single-base (T) insertions, and all mutations occurred on chromosomes 3B and 3D. Mutant lines... fus3 -8 and fus3 The -9 editing type remained unchanged in generations T4, T5, and T6, and was stably inherited on chromosomes 3B and 3D, indicating that the gene-edited lineage... fus3 -8 and fus3 -9 is already homozygous.

[0059] Table 6 (Transfer) TaFUS3 Sequence alignment results of gene-edited plants 3.2 Wheat TaFUS3 Validation of gene expression levels in overexpression and mutant lines 3.2.1 The development process of wheat grains TaFUS3 level of expression To clarify wheat TaFUS3 The effects of overexpression and gene editing on endogenous gene expression during grain development were investigated using overexpression and gene-edited lines and wild-type wheat grains at 15, 20, 25, 30, and 35 days post-flowering as materials, and quantitative real-time RT-PCR was performed. The results showed ( Picture 9 Compared to the wild type, the overexpression lines showed... TaFUS3 The expression level of the compound was significantly higher than that of the control throughout the entire grain development process, and the expression level showed a trend of first increasing and then decreasing as the grain development time was extended. TaFUS3 Gene expression differed among the three overexpression lines. OE-8 and OE-16 showed the highest expression levels at 25 days after flowering, with expression decreasing at 35 days, while OE-4 maintained a high expression level throughout the entire growth period. TaFUS3 The expression levels in the two gene-edited lines were significantly lower than those in the overexpression lines, and the expression levels were essentially similar to those in the wild type throughout development. TaFUS3 Overexpression of these genes leads to changes in the expression levels of endogenous genes, and TaFUS3 Gene editing did not affect TaFUS3 Gene transcription.

[0060] 3.2.2 The germination process of wheat grains TaFUS3 Expressive characteristics The results of the quantitative RT-PCR assay showed that ( Picture 10 During the 0-24 h period of grain swelling, TaFUS3 Gene expression showed a gradual upward trend, followed by a downward trend at 48 h. Compared to the wild type, throughout the entire grain germination process, TaFUS3 The expression levels in the overexpression lines were significantly higher than those in the wild-type and gene-edited lines. At 0, 12, 24, and 48 h of grain imbibition, TaFUS3 The expression levels in the overexpression lines were 79, 293, 165, and 350 times higher than those in the gene-edited lines and wild-type lines, respectively. TaFUS3 The expression level in the gene-edited lines was significantly lower than that in the overexpression lines. Although the expression levels at 12, 24 and 48 h of grain swelling were slightly higher than those in the wild type, the differences were not statistically significant.

[0061] 3.3 TaFUS3 Effects on starch and its component content in wheat grains Overexpressing, mutant, and wild-type wheat harvested at physiological maturity were left at room temperature for one month, and their starch content and composition were measured. The results (Table 7) showed that compared with the wild type, the total starch and amylopectin content of the overexpressing lines OE-4, OE-8, and OE-16 were significantly increased, with average increases of 32.40% and 37.47%, respectively. Except for OE-16, where no significant difference was observed, the amylose content of the overexpressing lines OE-4 and OE-8 was significantly higher than that of the wild type, increasing by 19.72% and 16.40%, respectively. Gene-edited lines... fus3-8 and fus3-9 The total starch and amylopectin content were both increased compared to the control, but the increase was slightly less than that of the overexpression line. These results indicate that... TaFUS3 Genes promote starch accumulation in wheat grains, which is beneficial for carbon metabolism and synthesis.

[0062] Table 7. Total starch, amylose, and amylopectin content Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. The application of B3 transcription factor TaFUS3, nucleic acid encoding TaFUS3, and biological material containing said nucleic acid in regulating starch content in wheat grains, characterized in that, The biological material is an expression cassette, transposon, plasmid vector, viral vector, or host cell; the amino acid sequence of TaFUS3 is shown in SEQ ID NO:

1.

2. The application according to claim 1, characterized in that, The sequence of the nucleic acid encoding TaFUS3 is shown in SEQ ID NO:

2.

3. The application of B3 transcription factor TaFUS3, nucleic acid encoding TaFUS3, and biological material containing said nucleic acid in cultivating wheat with increased grain starch content, characterized in that... The biological material is an expression cassette, transposon, plasmid vector, viral vector, or host cell; the amino acid sequence of TaFUS3 is shown in SEQ ID NO:

1.

4. The application according to claim 3, characterized in that, The sequence of the nucleic acid encoding TaFUS3 is shown in SEQ ID NO:

2.

5. A method for increasing the starch content in wheat grains, characterized in that, include: By increasing the content and / or activity of the B3 transcription factor TaFUS3 in recipient wheat, overexpressing wheat is obtained, wherein the starch content of the overexpressing wheat grains is higher than that of the recipient wheat; the amino acid sequence of TaFUS3 is shown in SEQ ID NO:

1.

6. The method for increasing the starch content in wheat grains according to claim 5, characterized in that, A method to increase the content and / or activity of the B3 transcription factor TaFUS3 in recipient wheat is to overexpress the TaFUS3 protein in recipient wheat; the overexpression method is to introduce a nucleic acid molecule encoding the TaFUS3 protein into recipient wheat.

7. The method for increasing the starch content in wheat grains according to claim 6, characterized in that, The overexpression method is as follows: the nucleic acid molecule encoding the TaFUS3 protein is introduced into the recipient wheat using Agrobacterium-mediated transformation.

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