Application of sorghum SbMyb1 gene in regulation and control of grain yield, tannin and anthocyanin

Abstract

The invention discloses application of a sorghum SbMyb1 gene to regulation and control of grain yield, tannin and anthocyanin, and relates to the field of plant genetic engineering. The invention specifically discloses an application of a SbMyb1 gene or a related biological material thereof in regulation and control of plant economic traits. Experiments prove that the SbMyb1 gene encoding the protein with the amino acid sequence shown as SEQ ID NO: 2 can negatively regulate and control yield-related traits such as grain length and grain weight and positively regulate and control the content of secondary metabolites such as grain tannin and anthocyanin, has a good application prospect in high-yield, high-quality and stress-resistant molecular design breeding, and has a wide application prospect. And a new research thought is provided for the SbMyb1 gene to participate in the grain development process through the function exertion of the MBW complex.

Description

Technical Field

[0001] The present invention relates to the field of plant genetic engineering, and in particular to the application of sorghum SbMyb1 gene in regulating grain yield, tannin and anthocyanin. Background Art

[0002] Sorghum is the world's fifth-largest crop, boasting excellent resistance to drought, flooding, salinity, and infertility. High seed yield is a primary goal of sorghum crop breeding. Crop seed yield is primarily determined by the number of ears per mu, the number of grains per spike, and 1000-grain weight. In sorghum, only a few yield-related genes have been reported, including SbqTGW1a and SbDEP1a, which control 1000-grain weight, and MSD1, MSD2, and MSD3, which control grain number per spike. Research on the molecular genetic basis of sorghum yield control is currently very limited, significantly limiting the molecular progress of high-yield sorghum breeding. Summary of the Invention

[0003] The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the present invention provides an application of the SbMyb1 gene or its related biological materials.

[0004] The present invention also provides a method for cultivating plants with increased or decreased grain length, grain width, 1000-grain weight, number of grains per ear, cell number of grain endosperm cells, total cell area of grain endosperm cells, tannins and / or anthocyanins.

[0005] The first aspect of the present invention provides the use of the SbMyb1 gene or its related biological material in any one of A1) to A9):

[0006] A1) Regulate plant grain length;

[0007] A2) regulating plant grain width;

[0008] A3) regulating plant thousand-grain weight;

[0009] A4) regulating the number of grains per ear of plants;

[0010] A5) Regulates the cell number of endosperm cells in plant grains;

[0011] A6) regulating the total cell area of endosperm cells in plant grains;

[0012] A7) regulating tannin synthesis in plants;

[0013] A8) Regulates anthocyanin synthesis in plants;

[0014] A9) Plant breeding;

[0015] The SbMyb1 gene encodes any one of the following proteins B1) to B3):

[0016] B1) a protein with an amino acid sequence as shown in SEQ ID NO: 2;

[0017] B2) a protein obtained by substituting and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID NO: 2;

[0018] B3) A fusion protein obtained by linking a tag to the N-terminus and / or C-terminus of the protein shown in B1) or B2).

[0019] In some embodiments of the present invention, the plant is a dicotyledonous plant or a monocotyledonous plant.

[0020] In some embodiments of the invention, the plant comprises a plant of the family Poaceae.

[0021] In some embodiments of the invention, the plant comprises a plant of the genus Sorghum.

[0022] In some embodiments of the invention, the plant comprises sorghum.

[0023] In some embodiments of the present invention, the protein can be artificially synthesized, or its encoding gene can be synthesized first and then biologically expressed.

[0024] In some embodiments of the present invention, the amino acid sequence of the protein in B2) is shown as SEQ ID NO: 17, SEQ ID NO: 19 or SEQ ID NO: 21.

[0025] In some embodiments of the present invention, the tag in B3) refers to a polypeptide or protein that is fused with the target protein using in vitro DNA recombination technology to facilitate expression, detection, tracing, and / or purification of the target protein. The tag may be a Flag tag, a His tag, an MBP tag, an HA tag, a myc tag, a GST tag, and / or a SUMO tag.

[0026] In some embodiments of the present invention, the biological material includes a substance for increasing or inhibiting the expression level of the gene encoding the protein or the activity or content of the protein.

[0027] In some embodiments of the present invention, the biomaterial comprises at least one of C1) to C7):

[0028] C1) a nucleic acid molecule encoding the protein;

[0029] C2) a nucleic acid molecule that increases the expression of the gene encoding the protein or the activity or content of the protein;

[0030] C3) a nucleic acid molecule that inhibits the expression of the gene encoding the protein or the activity or content of the protein;

[0031] C4) an expression cassette containing the nucleic acid molecule according to any one of C1) to C3);

[0032] C5) a recombinant vector containing the nucleic acid molecule of any one of C1) to C3) or the expression cassette of C4);

[0033] C6) a recombinant microorganism containing the nucleic acid molecule of any one of C1) to C3), the expression cassette of C4), or the recombinant vector of C5);

[0034] C7) A transgenic cell containing the nucleic acid molecule of any one of C1) to C3), the expression cassette of C4), or the recombinant vector of C5).

[0035] In some embodiments of the present invention, the nucleic acid molecule in any one of C1) to C3) may be DNA (such as cDNA, genomic DNA or recombinant DNA); the nucleic acid molecule may also be RNA (such as mRNA or hnRNA, etc.).

[0036] In some embodiments of the present invention, the nucleotide sequence of the nucleic acid molecule in C1) is as shown in any one of C11) to C13):

[0037] C11) a DNA molecule with a nucleotide sequence as shown in SEQ ID NO: 3, SEQ ID NO: 18, SEQ ID NO: 20 or SEQ ID NO: 22;

[0038] C12) a DNA molecule that has 80% or more identity with the nucleotide sequence shown in SEQ ID NO: 3, SEQ ID NO: 18, SEQ ID NO: 20, or SEQ ID NO: 22 and encodes a protein having the same function;

[0039] C13) A DNA molecule that hybridizes with the DNA molecule described in C11) or C12) under stringent conditions and encodes a protein having the same function. Taking into account the degeneracy of codons and the codon preferences of different species, those skilled in the art can use codons suitable for expression in a specific species as needed, as long as the resulting nucleic acid molecule can encode the protein.

[0040] Herein, identity refers to the identity of amino acid sequences or nucleotide sequences. The identity of amino acid sequences can be determined using homology search sites on the Internet, such as the BLAST page on the NCBI homepage.

[0041] In some embodiments of the present invention, the expression cassette in C4) refers to DNA capable of expressing the nucleic acid molecule in a host cell. The expression cassette may further include regulatory sequences. The regulatory sequences include, but are not limited to, a leader sequence (mRNA untranslated region sequence), a polyadenylation sequence, an enhancer sequence, a promoter, and a transcription terminator.

[0042] In some embodiments of the present invention, the vector in C5) may be a plasmid, cosmid, phage or viral vector.

[0043] In some embodiments of the present invention, the recombinant microorganism in C6) may be a fungus (eg, yeast) or a bacterium (eg, Escherichia coli).

[0044] In some embodiments of the present invention, the transgenic cells in C7) do not include any reproductive material.

[0045] In some embodiments of the present invention, the plant breeding described in A9) includes any one of A91) to A98):

[0046] A91) Cultivating plants with increased or decreased grain length;

[0047] A92) Cultivating plants with increased or decreased grain width;

[0048] A93) Cultivating plants with increased or decreased 1000-grain weight;

[0049] A94) Breeding plants with increased or decreased grain number per ear;

[0050] A95) Cultivating plants in which the cell number of endosperm cells in grains is increased or decreased;

[0051] A96) Cultivating plants in which the total cell area of endosperm cells in grains is increased or decreased;

[0052] A97) Breeding plants with increased or decreased tannin content;

[0053] A98) Breeding plants with increased or decreased anthocyanin content.

[0054] In some embodiments of the present invention, the plant breeding in A9) comprises:

[0055] Plants in which the expression level of the gene encoding the protein is increased or suppressed or the activity or content of the protein is hybridized with other plants for plant breeding.

[0056] In some embodiments of the present invention, the plant breeding in A9) comprises:

[0057] Detect the expression level of the protein or its encoding gene in the plant to be tested, and judge or assist in the regulation of grain length, grain width, thousand-grain weight, number of grains per ear, cell number of grain endosperm cells, total cell area of grain endosperm cells, tannins and / or anthocyanins of the plant to be tested.

[0058] In some embodiments of the present invention, determining or assisting in the regulation of grain length, grain width, thousand-grain weight, number of grains per ear, number of cells of grain endosperm cells, total cell area of grain endosperm cells, and tannins and / or anthocyanins of the plant to be tested includes:

[0059] If the plant to be tested expresses the protein with the amino acid sequence shown in SEQ ID NO:17, SEQ ID NO:19 and / or SEQ ID NO:21, the grain length, grain width, 1000-grain weight, cell number of grain endosperm cells and / or total cell area of grain endosperm cells of the plant to be tested are higher than those of the plant expressing the protein with the amino acid sequence shown in SEQ ID NO:2, and the tannin content and / or anthocyanin content are lower than those of the plant expressing the protein with the amino acid sequence shown in SEQ ID NO:2.

[0060] A second aspect of the present invention provides a method for cultivating plants with increased or decreased grain length, grain width, 1000-grain weight, number of grains per ear, number of cells in grain endosperm cells, total cell area of grain endosperm cells, tannins, and / or anthocyanins, the method comprising:

[0061] Increasing or inhibiting the expression level of the gene encoding the protein or the activity or content of the protein in the first embodiment of the target plant.

[0062] In some embodiments of the present invention, increasing the expression level of the gene encoding the protein in the target plant is achieved by introducing a nucleic acid molecule encoding the protein into the target plant.

[0063] In some embodiments of the present invention, the inhibiting the expression level of the gene encoding the protein in the target plant is achieved by knocking out or mutating the gene encoding the protein in the target rice plant.

[0064] In some embodiments of the present invention, the inhibition of the expression of the gene encoding the protein in the target plant is achieved by CRISPR / Cas9 technology.

[0065] In some embodiments of the present invention, the target sequence in the CRISPR / Cas9 technology is shown as SEQ ID NO:8 and SEQ ID NO:9.

[0066] In some embodiments of the present invention, the linker primer pair used in the CRISPR / Cas9 technology is shown as SEQ ID NO: 10 and SEQ ID NO: 11.

[0067] The beneficial effects of the present invention are:

[0068] The SbMyb1 gene is a negative regulatory factor in regulating yield-related traits such as grain length, width and weight, and a positive regulatory factor in regulating the content of secondary metabolites such as tannins and anthocyanins in grains. It has great application prospects in molecular design breeding for high yield, high quality and stress resistance, and also provides new research ideas for the SbMyb1 gene to participate in the grain development process through the MBW complex.

[0069] Other features and advantages of the present invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0070] Figure 1 Schematic diagram of the structure of the recombinant expression vector pCAMBIA2300-Myc-SbMyb1;

[0071] Figure 2 Schematic diagram of the mutation status of different SbMyb1 gene editing mutant lines; A: P226, B: P184;

[0072] Figure 3 The results of SbMyb1 gene expression level detection in different SbMyb1 gene overexpression lines and SbMyb1 gene editing mutant lines under the P226 background are shown;

[0073] Figure 4 Statistical results of grain yield-related traits of SbMyb1 gene overexpression lines and SbMyb1 gene editing mutant lines under the P226 background; A: Comparison of mature grain length (scale: 2 cm), B: Comparison of mature grain width (scale: 2 cm), C: Statistical results of grain length, D: Statistical results of grain width, E: 1000-grain weight, F: Statistical results of grain number per spike; * indicates significant difference (P<0.05), ** indicates extremely significant difference (P<0.01), *** indicates extremely significant difference (P<0.001);

[0074] Figure 5Statistical results of grain yield-related traits of SbMyb1 gene-edited mutant lines under the P184 background; A: Comparison of mature grain length and width (scale: 2 cm), B: Grain length statistics, C: Grain width statistics, D: 1000-grain weight statistics; * indicates significant differences (P<0.05), ** indicates extremely significant differences (P<0.01);

[0075] Figure 6 Figure 2 shows the morphological characteristics of endosperm cells in the SbMyb1 gene overexpression line and the SbMyb1 gene editing mutant line during the grain filling stage under the P226 background; A: Paraffin section scanning electron microscopy of sorghum kernel cross-section at the grain filling stage (scale bar: 200 μm), B: Cell number, C: Single cell area, D: Total cell area; * indicates significant difference (P < 0.05), ** indicates extremely significant difference (P < 0.01);

[0076] Figure 7 These are the total tannin and total anthocyanin detection results of grains of different SbMyb1 gene overexpression lines and SbMyb1 gene-edited mutant lines; A: tannin content of grains of SbMyb1 gene-edited mutant lines under the P184 background, B: anthocyanin content of grains of SbMyb1 gene-edited mutant lines under the P226 background, C: anthocyanin content of grains of SbMyb1 gene-edited mutant lines under the P184 background; * indicates significant difference (P<0.05), *** indicates extremely significant difference (P<0.001). DETAILED DESCRIPTION

[0077] The following will clearly and completely describe the concept and technical effects of the present invention in conjunction with the embodiments to fully understand the purpose, features and effects of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0078] If the specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or those recommended by the manufacturer. All reagents or instruments used, if the manufacturer is not specified, are commercially available conventional products.

[0079] Unless otherwise specified, "room temperature" in the present invention means (25±5)°C.

[0080] The genomic sequence of the SbMyb1 gene is shown in SEQ ID NO: 1, and the amino acid sequence of the Myb1 protein encoded thereby is shown in SEQ ID NO: 2.

[0081]

[0082] MGRKPCCPKEGLNRGAWTSMEDDILVSYIQKHGEGKWGSLPRRAGLKRCGKSCRLRWLNYLRPGIKRGNISDDEEELIIRLHRLLGNRWSLIAGRLPGRTDNEIKNYWNTTLGKKVLKNSGGLKEDSEAAGASSKRRPFSEPTRTVSDT AAQAHARGRPPEPPEASLVRSKALRCTRTAMMLPVAQPTAHARHGRALETAAGDDHVKAEQTVVVVKAPTVEVRQDHLPEDDLPIDIDLDFDMGEMGFLSPWRGEVADGIEPGGQFGGGELDDLEALLLGPGGDDDVHEFAWF*(SEQ ID NO:2).

[0083] Example 1 (Construction of SbMyb1 gene overexpression strain)

[0084] 1. Construction of recombinant expression vector pCAMBIA2300-Myc-SbMyb1:

[0085] The CDS sequence of the SbMyb1 gene (as shown in SEQ ID NO: 3) was inserted into the target plant expression vector pCAMBIA2300-Ubi-Myc (preserved by this laboratory, this vector is based on the commercial pCAMBIA2300 vector and has an Ubi promoter and OCS terminator added, and contains a Myc tag sequence. The public can request this vector from the applicant for use in repeated experiments of the present invention) by homologous recombination.

[0086] ATGGGGAGGAAGCCATGCTGCCCCAAGGAGGGGCTGAACAGGGGAGCTTGGACATCAATGGAGGATGACATCCTTGTCTCCTACATCCAGAAGCATGGAGAGGGAAAATGGGGAAGCCTCCCCAGAAGAGCTGGACTGAAGCGGTGTGGGAAGAGCTGCCGGCTGCGGTGGCTCAACTACCTCCGGCCGGGTATCAAGCGAGGTAACATCTCGGATGATGAAGAGGAGCTCATCATTAGGCTCCACAGGCTCCTAGGCAACAGGTGGTCGCTGATCGCCGGGCGGCTGCCGGGGCGAACAGACAATGAAATCAAGAACTACTGGAACACGACGCTCGGCAAGAAGGTGCTCAAGAACAGCGGCGGCTTGAAAGAAGACAGCGAGGCCGCCGGCGCATCATCGAAACGCAGGCCATTCTCAGAACCGACCAGAACCGTCAGCGACACTGCCGCCCAGGCTCATGCGCGTGGGCGGCCGCCGGAGCCACCGGAGGCAAGCCTGGTCCGGAGCAAGGCGCTGCGTTGCACCAGAACTGCCATGATGCTGCCGGTGGCGCAGCCCACCGCCCATGCGCGCCACGGCCGTGCGTTGGAGACAGCTGCGGGCGACGATCACGTTAAGGCGGAGCAGACGGTCGTCGTCGTCAAGGCGCCAACTGTGGAGGTGCGACAGGATCATCTGCCGGAGGATGACTTGCCGATCGACATCGACCTCGACCTCGACTTCGACATGGGTGAGATGGGTTTCCTGAGCCCGTGGCGCGGCGAGGTCGCTGACGGCATAGAGCCAGGCGGTCAGTTCGGCGGTGGCGAGCTTGACGATCTGGAGGCGCTGCTGCTGGGGCCCGGAGGTGACGATGATGTTCATGAGTTTGCGTGGTTCTGA(SEQ ID NO:3).

[0087] The steps are as follows:

[0088] (1) RNA from sorghum (variety P184) was extracted using an ultrapure RNA extraction kit (Beijing Kangwei Century Biotechnology Co., Ltd., catalog number: CW0581). The RNA was reverse transcribed into cDNA using a Fast Quant reverse transcription kit (Tiangen Biochemical Technology (Beijing) Co., Ltd.). PCR amplification was performed using the cDNA as a template, and the CDS sequence of the SbMyb1 gene as shown in SEQ ID NO: 3 was obtained by sequencing.

[0089] (2) Synthesize the adapter primers Myb1-JT-F and Myb1-JT-R for homologous recombination, which are located near the restriction sites SpeI and BamHI, respectively. PCR amplification was performed using the adapter primers using the CDS sequence of the SbMyb1 gene as a template. The amplified product was gel-recovered using a Promega gel recovery kit (Cat. No. ADA9298) to obtain a purified amplified product (the CDS sequence of the SbMyb1 gene containing the adapter sequence).

[0090] Myb1-JT-F: gaggacttgaattcgactagtATGGGGAGGAAGCCATGCTG (SEQ ID NO: 4);

[0091] Myb1-JT-R: caggtcgactctagaggatccTCAGAACCACGCAAACTCAT (SEQ ID NO: 5).

[0092] (3) The vector pCAMBIA2300-Ubi-Myc was double-digested with restriction endonucleases SpeI and BamHI (purchased from NEB), and the digested product was recovered by gel extraction using a Promega gel extraction kit (Cat. No. ADA9298) to obtain the digested vector.

[0093] The enzyme digestion system is: 3 μL of 10× Cutsmart buffer, 1 μL of SpeI, 1 μL of BamHI, 2 μg of vector, and ddH2O to 30 μL.

[0094] The enzyme digestion conditions were: reaction at 37°C for 2 hours.

[0095] (4) A ligase-independent single-fragment one-step cloning kit (purchased from Nanjing Novozymes Biotechnology Co., Ltd., catalog number: C112-01) was used to perform homologous recombination on the purified amplified product and the enzyme-digested vector.

[0096] Mix the purified amplified product and digested vector at a 2:1 molar ratio. Add 2 μL of 5× CEII Buffer and 1 μL of Exnase II, and make up to 10 μL with ddH2O. Mix thoroughly and incubate at 37°C for 30 minutes. After completion, immediately cool the recombinant product on ice and store at -20°C.

[0097] (5) Add 10 μL of the recombinant product to DH5α E. coli competent cells that have been melted on ice. After mixing, place them on ice for 30 minutes, heat shock at 42°C for 90 seconds, and immediately place them on ice for 2-3 minutes. Heat shock at 42°C for 30 seconds, and quickly place them on ice for 2 minutes. Then add 500 μL of LB medium without antibiotics, culture at 37°C with shaking for 1 hour (shaking speed is 220 rpm), centrifuge at 5,000 rpm for 1 minute, discard the supernatant, resuspend the precipitate in LB medium, spread it on LB agar plates containing the corresponding antibiotics, and culture at 37°C for 12h-16h. Pick 1-5 monoclonal colonies for PCR verification, and positive clones are identified by agarose gel electrophoresis and sequencing.

[0098] Add 10 μL of sterile water to a 96-well 0.2 mL PCR plate, gently pick up a single colony with a white pipette tip and transfer it to the 96-well plate, gently pipetting until the colony is completely dissolved. 8 μL of bacterial solution was inoculated into 500 μL of liquid culture medium with antibiotics and cultured with shaking at 37°C for 2h-3h; the remaining 2 μL of bacterial solution was used as a PCR template and amplified using Taq enzyme (Kangwei Century) (forward primer 2300-Ubi-F: TCGATGCTCACCCTGTTGTT (SEQ ID NO: 6); reverse primer 2300-Ocs-R: ATCATAGGCGTCTCGCATATCTC (SEQ ID NO: 7). 200 μL of the corresponding bacterial solution identified as positive by PCR was sent to the company for sequencing; the remaining 300 μL of bacterial solution was inoculated into 5 mL of liquid LB culture medium with antibiotics and cultured with shaking at 37°C overnight. According to the sequencing results, 5 mL of the corresponding bacterial solution verified to be correct by sequencing was inoculated into 500 mL of liquid LB culture medium with antibiotics and cultured with shaking at 37°C overnight.

[0099] It was verified that the SbMyb1 gene was successfully connected to the pCAMBIA2300-Ubi-Myc vector to obtain the Myc-tagged overexpression fusion vector pCAMBIA2300-Myc-SbMyb1, which can express the Myc-tagged SbMyb1 protein (Myc-Myb1).

[0100] 2. Acquisition of SbMyb1 gene overexpression strains:

[0101] (1) A single colony verified by PCR and sequencing was inoculated into 5 mL of LB liquid medium containing the corresponding antibiotics and cultured at 37°C with shaking for 12-14 hours at a shaking speed of 220 rpm. The bacterial solution was centrifuged at 12,000 rpm for 30 seconds, the bacterial solution was collected, the supernatant was discarded, 250 μL of P1 buffer was added, and the solution was vortexed to precipitate the bacterial solution. Then, 250 μL of P2 buffer was added and the solution was gently inverted 4-6 times to clarify the bacterial solution (this step should not exceed 5 minutes). Subsequently, 350 μL of N3 buffer was added, the solution was gently inverted to mix, and the solution was centrifuged at 12,000 rpm for 5 minutes. The supernatant was transferred to the adsorption column. The solution was centrifuged at 12,000 rpm for 30 seconds, the filtrate was discarded, and 150 μL of PB buffer was added to the adsorption column, the solution was centrifuged at 12,000 rpm for 30 seconds, and the filtrate was discarded. 700 μL of wash buffer was added to the adsorption column, the solution was centrifuged at 12,000 rpm for 1 minute, and the filtrate was discarded. Repeat this step once, washing the column with 500 μL of wash buffer. Finally, return the column to a fresh centrifuge tube and centrifuge at 12,000 rpm for 2 minutes to remove residual liquid. Transfer the column to a new 1.5 mL centrifuge tube, add 30 μL of distilled water, and centrifuge at 12,000 rpm for 1 minute to elute the plasmid. Determine the plasmid concentration using a NanoDrop 2000 and store the plasmid at -20°C until use.

[0102] (2) Electrotransformation of Agrobacterium competent cells:

[0103] Thaw competent Agrobacterium cells on ice, add the plasmid to be transformed, mix gently, and incubate on ice for 30 minutes. After electroporation (electroporation conditions: 1.8 kV, 5.6 ms), transfer the cells to a 1.5 mL centrifuge tube and add 500 μL of antibiotic-free LB medium. Incubate at 28°C with shaking for 2-3 hours at 200 rpm. After incubation, spread 30 μL of the bacterial solution onto solid LB medium containing antibiotics, invert the plate, and incubate at 28°C for 2 days. Positive colonies were selected and amplified by PCR using primers 2300-Ubi-F and 2300-Ocs-R. The PCR product was approximately 1492 bp in size. The positive strain identified by PCR was designated recombinant Agrobacterium EHA105 / pCAMBIA2300-Myc-SbMyb1 and stored at -80°C.

[0104] (3) The recombinant Agrobacterium EHA105 / pCAMBIA2300-Myc-SbMyb1 was transformed into the sorghum recipient material P226 through Agrobacterium-mediated method.

[0105] 1) Plant P226 sorghum plants in a greenhouse. When the plants reach the 15th day of flowering, cut the ears and squeeze out as many immature embryos as possible from the grain-filled kernels in a sterile laminar flow hood. Place the embryos evenly on callus culture medium for callus culture.

[0106] 2) The recombinant Agrobacterium EHA105 / pCAMBIA2300-Myc-SbMyb1 was streaked onto YEP solid medium supplemented with Kan, and the resulting single colony was inoculated into 10 mL of YEP liquid medium supplemented with Kan for primary activation (28°C, 220 rpm, shaking overnight). The activated bacterial solution was inoculated into 80 mL of YEP liquid medium supplemented with Kan at a ratio of 1:1000 (V:V), and cultured at 28°C, 220 rpm, shaking until the OD 600nm is 0.8-1.0, and a recombinant Agrobacterium liquid is obtained.

[0107] 3) Infection:

[0108] The recombinant Agrobacterium bacterial solution was transferred to a 50 mL centrifuge tube and centrifuged at 5000 rpm for 10 min to enrich the bacteria. The bacteria were then fully resuspended with a resuspension buffer, added to the callus tissue, and infected and transformed. The infected callus tissue was transferred to a rooting medium (containing NPT II) for rooting culture. If the culture medium browned, the rooting medium needed to be transferred multiple times. The infected sorghum plants were cultured normally until maturity, and their mature seeds (i.e., T0 generation transgenic seeds) were harvested. The SbMyb1 gene overexpression strains SbMyb1-OE-1 and SbMyb1-OE-2 corresponding to P226 were obtained.

[0109] Example 2 (Construction of SbMyb1 gene knockout strain)

[0110] (1) Enzyme digestion of the vector:

[0111] The CmYLCV-pBUE411 vector (provided by Xie Qi's group at the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences) was digested with BsaI-HFv2 (NEB) and isolated by gel recovery. The digested vector had specific sticky ends, ACGT and GTTT.

[0112] The enzyme digestion system is: 2 μg plasmid, 1 μL BsaI-HFv2 enzyme, and ddH2O is added to 30 μL.

[0113] The enzyme digestion conditions were: reaction at 37°C for 2 hours.

[0114] (2) The gene editing system developed by Liu Yaoguang's laboratory at South China Agricultural University was used. Targets were found using the scores from the CRISPOR website (http: / / crispor.tefor.net / crispor.py), and off-target sites were found using the Cas-OFFinder website (http: / / www.rgenome.net / cas-offinder / ). The target was required to have a high score (generally >45 points, no mismatches within two bases, and preferably fewer mismatches within three bases). If enzyme identification was required, it was best if the Cas9 cleavage site was on the endonuclease recognition sequence.

[0115] For the target sequences CAGGGGAGCTTGGACATCAA (SEQ ID NO: 8) and CTCCATGCTTCTGGATGTAG (SEQ ID NO: 9), a forward primer containing a TGCA linker (ATATATggtctcatgcaCA GGGGAGCTTGGACATCAAGTTTTAGAGCTAGAAATAGC (SEQ ID NO: 10)) and a reverse primer containing an AAAC linker (ATATATGGTCTCaAAACCTCCATGCTTCTGGATGTAGtgcaccagccggg aatcgaa (SEQ ID NO: 11)) were synthesized. After primer annealing, the target sequence was ligated into the CmYLCV-pBUE411 vector treated with Bsa I using T4 DNA ligase to generate the vector CmYLCV-pBUE411-SbMyb1.

[0116] (3) Amplification and ligation of gene editing target sequence fragments:

[0117] Using the CmYLCV-pBUE411-SbMyb1 vector as a template, KOD One PCR Master Mix (Cat. No. KMM-201) was used to amplify the fragment containing the gene editing target sequence (cgattcccggctggtgcaCAGGGGAGCTTGGACATCAAGTTTTAGAGCTAGAAATAGCAAGTTAA The protein sequence of the protein AATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCGCATCTAGgcaccagtggtctagtggtagaatagtaccctgccacggtacagacccgggttcgattcccggctggtgcaCTACATCCAGAAGCATGGAGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCGCATCTAGgcaccagtggtctagtggtagaatagtaccctgccacggtacagacccgggttcgattcccggctggtgca (SEQ ID NO: 12) was recovered by gel extraction using a Promega gel extraction kit to obtain a 220 bp product, which was then digested with BsaI.

[0118] A 10 μL ligation system (1 μL of 10×T4 DNA ligase Buffer, 2 μL of CmYLCV-pBUE411 vector digested with BsaI, 1 μL of BsaI digested, 5 μL of gene editing target sequence fragment digested with BsaI, 1 μL of T4 DNA Ligase, and 1 μL of ddH2O) was ligated at 37°C for 20 min to obtain a ligation product.

[0119] (4) Add 10 μL of the ligation product obtained in step (3) to DH5α E. coli competent cells that have been melted on ice. After mixing, place them on ice for 30 minutes, heat shock them at 42°C for 90 seconds, and immediately place them on ice for 2-3 minutes. Heat shock them at 42°C for 30 seconds and quickly place them on ice for 2 minutes. Then add 500 μL of LB medium without antibiotics, culture them at 37°C with shaking for 1 hour (shaking speed is 220 rpm), centrifuge them at 5,000 rpm for 1 minute, discard the supernatant, resuspend the precipitate in LB medium, spread it on LB agar plates containing the corresponding antibiotics, and culture them at 37°C for 12h-16h. Pick 1-5 monoclonal colonies for PCR verification, and positive clones are identified by agarose gel electrophoresis and sequencing.

[0120] Add 10 μL of sterile water to a 96-well 0.2 mL PCR plate. Gently pick up a single colony with a white pipette tip and transfer it to the 96-well plate. Gently pipette until the colony is completely dissolved. Take 8 μL of the bacterial solution and inoculate it into 500 μL of liquid culture medium with antibiotics. Incubate with shaking at 37°C for 2-3 hours. The remaining 2 μL of bacterial solution is used as a PCR template and amplified using Taq enzyme (Cornwell). (Forward primer CmYLCV-SEQ: GAAGTATTCAGGCAGGTGGC (SEQ ID NO: 13), reverse primer CmYLCV-R: TGACACACCAAATATTTCATCT (SEQ ID NO: 14). After inserting the two target sites, the product size is 531 bp, and the original vector is 356 bp.)

[0121] The specific target site was sequenced using primers SbMyb1-cas-F (GATGCAAGAGCATCCGATCT (SEQ ID NO: 15)) and SbMyb1-cas-R (CATGTCATGCTTGCGCATTG (SEQ ID NO: 16)). The target band size was 643 bp. The positive clones obtained were verified to contain the successfully recombined pYLCRISPR / Cas9P ubi -B-Myb1 plasmid.

[0122] 2. Obtaining SbMyb1 gene-edited mutant strains:

[0123] (1) Verify the correct pYLCRISPR / Cas9P ubi -B-Myb1 plasmid was electroporated into Agrobacterium competent cells (same steps as in Example 1).

[0124] PCR identification was performed using primers SbMyb1-cas-F and SbMyb1-cas-R. The positive Agrobacterium identified by PCR was named recombinant Agrobacterium EHA105 / pYLCRISPR / Cas9P ubi -B-Myb1, stored at -80°C.

[0125] (2) Recombinant Agrobacterium EHA105 / pYLCRISPR / Cas9P ubi -B-Myb1 was transferred into sorghum recipient materials P226 and P184 by Agrobacterium-mediated method (the steps were the same as those in Example 1). The mature seeds were harvested. ubi-B-Myb1 gene-edited plants were PCR amplified and sequenced for the target genomic position of the Myb1 gene using the forward primer SbMyb1-cas-F and the reverse primer SbMyb1-cas-R. The heterozygous materials with base insertion or deletion (frameshift mutation) in the target sequence were selected (the target gene target position of the gene-edited T0 generation positive plants were all chimeras) and continued to self-pollinate to the next generation to the T1 generation. Among the T1 generation segregated individuals, the primers SbMyb1-cas-F and SbMyb1-cas-R were used to continue to detect whether the Myb1 gene target position with frameshift mutation was homozygous. At the same time, the vector primers CmYLCV-SEQ and CmYLCV-R were used to detect whether the vector pYLCRISPR / Cas9P ubi -B-Myb1 is separated. The final vector pYLCRISPR / Cas9P was selected ubi -B-Myb1 was separated and the T1 generation homozygous mutant material with the sequence mutated at the target site of the Myb1 gene was homozygous for subsequent studies.

[0126] The comparison results of the target sequencing sequences of Myb1 gene editing mutant strains and Myb1 wild-type receptor materials P226 and P184 are shown in the figure. Figure 2 shown.

[0127] In the P226 background, the SbMyb1-KO (P226) plant lost a 41bp sequence in the corresponding region of the Myb1 genome (nucleotides 500 to 540 of SEQ ID NO:1), and caused a partial deletion in the corresponding region of the Myb1 protein (a stop codon TAA was prematurely generated at amino acid position 55 of SEQ ID NO:2, resulting in the premature termination of subsequent translation), thereby knocking out the Myb1 gene. The amino acid sequence of the encoded Myb1 protein is shown in SEQ ID NO:17, and the corresponding Myb1 genomic sequence is shown in SEQ ID NO:18.

[0128] Two mutant types were obtained in the P184 background, designated SbMyb1-KO-1(P184) and SbMyb1-KO-2(P184). The SbMyb1-KO-1(P184) plant lost 42 bp of sequence in the corresponding region of the Myb1 genome (nucleotides 500 to 541 of SEQ ID NO:1) and resulted in a partial deletion in the corresponding region of the Myb1 protein (prematurely generating the stop codon TGA at amino acid position 19 of SEQ ID NO:2, leading to the premature termination of subsequent translation). This knocked out the Myb1 gene. The amino acid sequence of the encoded Myb1 protein is shown in SEQ ID NO:19, and the corresponding Myb1 genomic sequence is shown in SEQ ID NO:20. The SbMyb1-KO-2 (P184) plant has a T inserted into the corresponding region of the Myb1 genome (nucleotide 56 of SEQ ID NO:1), and causes a partial deletion in the corresponding region of the Myb1 protein (premature generation of the stop codon TGA at amino acid position 23 of SEQ ID NO:2, resulting in the premature termination of subsequent translation), thereby knocking out the Myb1 gene. The amino acid sequence of the encoded Myb1 protein is shown in SEQ ID NO:21, and the corresponding Myb1 genomic sequence is shown in SEQ ID NO:22.

[0129] MGRKPCCPKEGLNRGAWTWRGKMGKPPQKSWTEAVWEELPAAVAQLPPAGYQAR*(SEQ ID NO:17);

[0130] ATGGGGAGGAAGCCATGCTGCCCCAAGGAGGGGCTGAACAGGGGAGCTTGGACATGGAGAGGGAAAATGGGGAAGCCTCCCCAGAAGAGCTGGACTGAAGCGGTGTGGGAAGAGCTGCCGGCTGCGGTGGCTCAACTACCTCCGGCCGGGTATCAAGCGAGGTAACATCTCGGATGATGAAGAGGAGCTCATCATTAGGCTCCACAGGCTCCTAGGCAACAGGTGGTCGCTGATCGCCGGGCGGCTGCCGGGGCGAACAGACAATGAAATCAAGAACTACTGGAACACGACGCTCGGCAAGAAGGTGCTCAAGAACAGCGGCGGCTTGAAAGAAGACAGCGAGGCCGCCGGCGCATCATCGAAACGCAGGCCATTCTCAGAACCGACCAGAACCGTCAGCGACACTGCCGCCCAGGCTCATGCGCGTGGGCGGCCGCCGGAGCCACCGGAGGCAAGCCTGGTCCGGAGCAAGGCGCTGCGTTGCACCAGAACTGCCATGATGCTGCCGGTGGCGCAGCCCACCGCCCATGCGCGCCACGGCCGTGCGTTGGAGACAGCTGCGGGCGACGATCACGTTAAGGCGGAGCAGACGGTCGTCGTCGTCAAGGCGCCAACTGTGGAGGTGCGACAGGATCATCTGCCGGAGGATGACTTGCCGATCGACATCGACCTCGACCTCGACTTCGACATGGGTGAGATGGGTTTCCTGAGCCCGTGGCGCGGCGAGGTCGCTGACGGCATAGAGCCAGGCGGTCAGTTCGGCGGTGGCGAGCTTGACGATCTGGAGGCGCTGCTGCTGGGGCCCGGAGGTGACGATGATGTTCATGAGTTTGCGTGGTTCTGA(SEQ ID NO:18);

[0131] MGRKPCCPKEGLNRGAWT*(SEQ ID NO:19);

[0132] ATGGGGAGGAAGCCATGCTGCCCCAAGGAGGGGCTGAACAGGGGAGCTTGGACATGAGAGGGAAAATGGGGAAGCCTCCCCAGAAGAGCTGGACTGAAGCGGTGTGGGAAGAGCTGCCGGCTGCGGTGGCTCAACTACCTCCGGCCGGGTATCAAGCGAGGTAACATCTCGGATGATGAAGAGGAGCTCATCATTAGGCTCCACAGGCTCCTAGGCAACAGGTGGTCGCTGATCGCCGGGCGGCTGCCGGGGCGAACAGACAATGAAATCAAGAACTACTGGAACACGACGCTCGGCAAGAAGGTGCTCAAGAACAGCGGCGGCTTGAAAGAAGACAGCGAGGCCGCCGGCGCATCATCGAAACGCAGGCCATTCTCAGAACCGACCAGAACCGTCAGCGACACTGCCGCCCAGGCTCATGCGCGTGGGCGGCCGCCGGAGCCACCGGAGGCAAGCCTGGTCCGGAGCAAGGCGCTGCGTTGCACCAGAACTGCCATGATGCTGCCGGTGGCGCAGCCCACCGCCCATGCGCGCCACGGCCGTGCGTTGGAGACAGCTGCGGGCGACGATCACGTTAAGGCGGAGCAGACGGTCGTCGTCGTCAAGGCGCCAACTGTGGAGGTGCGACAGGATCATCTGCCGGAGdGATGACTTGCCGATCGACATCGACCTCGACCTCGACTTCGACATGGGTGAGATGGGTTTCCTGAGCCCGTGGCGCGGCGAGGTCGCTGACGGCATAGAGCCAGGCGGTCAGTTCGGCGGTGGCGAGCTTGACGATCTGGAGGCGCTGCTGCTGGGGCCCGGAGGTGACGATGATGTTCATGAGTTTGCGTGGTTCTGA(SEQ ID NO:20);

[0133] MGRKPCCPKEGLNRGAWTFNGG*(SEQ ID NO:21);

[0134] ATGGGGAGGAAGCCATGCTGCCCCAAGGAGGGGCTGAACAGGGGAGCTTGGACATTCAATGGAGGATGACATCCTTGTCTCCTACATCCAGAAGCATGGAGAGGGAAAATGGGGAAGCCTCCCCAGAAGAGCTGGACTGAAGCGGTGTGGGAAGAGCTGCCGGCTGCGGTGGCTCAACTACCTCCGGCCGGGTATCAAGCGAGGTAACATCTCGGATGATGAAGAGGAGCTCATCATTAGGCTCCACAGGCTCCTAGGCAACAGGTGGTCGCTGATCGCCGGGCGGCTGCCGGGGCGAACAGACAATGAAATCAAGAACTACTGGAACACGACGCTCGGCAAGAAGGTGCTCAAGAACAGCGGCGGCTTGAAAGAAGACAGCGAGGCCGCCGGCGCATCATCGAAACGCAGGCCATTCTCAGAACCGACCAGAACCGTCAGCGACACTGCCGCCCAGGCTCATGCGCGTGGGCGGCCGCCGGAGCCACCGGAGGCAAGCCTGGTCCGGAGCAAGGCGCTGCGTTGCACCAGAACTGCCATGATGCTGCCGGTGGCGCAGCCCACCGCCCATGCGCGCCACGGCCGTGCGTTGGAGACAGCTGCGGGCGACGATCACGTTAAGGCGGAGCAGACGGTCGTCGTCGTCAAGGCGCCAACTGTGGAGGTGCGACAGGATCATCTGCCGGAGGATGACTTGCCGATCGACATCGACCTCGACCTCGACTTCGACATGGGTGAGATGGGTTTCCTGAGCCCGTGGCGCGGCGAGGTCGCTGACGGCATAGAGCCAGGCGGTCAGTTCGGCGGTGGCGAGCTTGACGATCTGGAGGCGCTGCTGCTGGGGCCCGGAGGTGACGATGATGTTCATGAGTTTGCGTGGTTCTGA(SEQ ID NO:22).

[0135] Detection Example 1

[0136] The Talent qPCR PreMix kit (Tiangen Biochemical Technology (Beijing) Co., Ltd., Cat. No. FP209) was used to detect the expression level of the SbMyb1 gene in each strain. The EIF gene was used as the internal reference gene for relative quantification. The detection steps were carried out strictly according to the instructions. The primers used to amplify the SbMyb1 gene were Myb1-qRT-seq-2F and Myb1-qRT-seq-2R; the primers used to amplify the EIF gene were EIF-qF and EIF-qR.

[0137] Myb1-qRT-seq-2F:ATGGGGAGGAAGCCATGCTG (SEQ ID NO: 23);

[0138] Myb1-qRT-seq-2R: TCAGAACCACGCAAACTCATG (SEQ ID NO: 24);

[0139] EIF-qF: CAACTTTGTCACCCGCGATGA (SEQ ID NO: 25);

[0140] EIF-qR:TCCAGAAACCTTAGCAGCCCA (SEQ ID NO:26).

[0141] Test results such as Figure 3 shown.

[0142] The results of qPCR showed that the expression levels of the SbMyb1 gene in SbMyb1-OE-1 and SbMyb1-OE-2 were between 16 and 24 times higher than that in the wild-type variety P226.

[0143] Test Example 2

[0144] Grain yield-related traits were analyzed for wild-type, SbMyb1 gene-overexpressing, and SbMyb1 gene-edited mutant lines. Paraffin sections and scanning electron microscopy (SEM) were used to observe and analyze the morphological characteristics of endosperm cells during the grain filling stage of wild-type P226, its corresponding SbMyb1 gene-edited mutant lines, and SbMyb1 gene-overexpressing lines to assess the cytological basis for the differences in grain size. The specific procedures for preparing paraffin sections are as follows:

[0145] 1) Place fresh seeds in FAA fixative (50 (v / v)% ethanol, 5 (v / v)% acetic acid, 3.7 (v / v)% formaldehyde, and the balance water) and slowly pump air for 15 minutes using a vacuum pump. Change the fixative once and allow the material in the fixative to be induced overnight at 4°C.

[0146] 2) Remove the fixed material and induce it with 50 (v / v)% ethanol-water solution at room temperature for 30 minutes. Repeat once. Then, induce it with 60 (v / v)% ethanol-water solution, 70 (v / v)% ethanol-water solution, and 85 (v / v)% ethanol-water solution at 4°C for 30 minutes each. Then, transfer the material to 95 (v / v)% ethanol-water solution containing 0.1% eosin and incubate at 4°C overnight. All subsequent operations were performed at 4°C unless otherwise specified.

[0147] 3) Transfer the material to anhydrous ethanol and incubate for at least 1 hour, then transfer to a fresh volume of anhydrous ethanol and continue incubating for 30 minutes. Repeat twice. Continue incubating in solutions with a xylene:ethanol volume ratio of 1 / 4:3 / 4, 1 / 2:1 / 2, and 3 / 4:1 / 4 for 30 minutes each. Discard the solution and replace with pure xylene. Incubate at room temperature for 1 hour, repeat twice. Finally, add a xylene-paraffin mixture (xylene:paraffin (v / v) = 1:1) and incubate at 4°C overnight.

[0148] 4) Place the mixture from step 3) in a 42°C incubator and incubate for 1 hour. Gradually add the paraffin wax fragments, each time incubating for 30 minutes. Incubate at 60°C for at least 4 hours. Repeat this process 4 to 6 times to fully melt the paraffin. Pour the mixture into an embedding tank placed on a warming table. Use a preheated dissecting needle to gently adjust the material to its correct position. Rapidly cool the mixture in a pre-prepared water tank. Store the embedded wax blocks in a 4°C refrigerator until ready for use.

[0149] 5) Secure the successfully embedded paraffin block to an automatic microtome and slice to a thickness of 6-8 μm. Wash the slides in ethanol, air-dry them, and add 3 μL of POLY-L-LYSINE (1 mg / mL) to each slide. Cover with a coverslip, press firmly, and dry at 42°C overnight.

[0150] 6) Slides were stained using safranin staining, using the following reagents in sequence: xylene (20 min), xylene (15 min), 1 / 2 xylene: 1 / 2 anhydrous ethanol (1-5 min), anhydrous ethanol (2 min), anhydrous ethanol (2 min), 95% ethanol (1 min), 85% ethanol (1 min), 70% ethanol (1 min), 50% ethanol (1 min), distilled water (1 min), safranin (12 h), 70% ethanol (30 s), 85% ethanol (30 s), 95% ethanol (30 s), 100% ethanol (1 min), 100% ethanol (1 min), xylene (3 min), and xylene (15 min). Finally, sections were examined under a microscope and mounted with Canada gum.

[0151] The results are as follows Figure 4 、 Figure 5 and Figure 6 shown.

[0152] In the P226 background, compared with P226, the grain length, width, and 1000-grain weight of the SbMyb1 gene-edited mutant strain were significantly increased, while the grain length, width, and 1000-grain weight of the SbMyb1 gene-overexpressing strain were significantly decreased. The cell number and total cell area of endosperm cells during the grain filling stage were significantly increased in the SbMyb1 gene-edited mutant strain, while the cell number, single cell area, and total cell area of the SbMyb1 gene-overexpressing strain were significantly decreased. This suggests that the SbMyb1 gene may affect grain development by regulating the expression of genes related to cell expansion.

[0153] In the P184 background, the grain length, grain width and 1000-grain weight of SbMyb1-KO-1 and SbMyb1-KO-2 were significantly increased compared with P184.

[0154] Furthermore, the SbMyb1 gene also affects the agronomic traits of sorghum ear length and grain number per ear. This further demonstrates the multiple regulatory functions of the SbMyb1 gene in sorghum grain development. These results suggest that the SbMyb1 gene negatively regulates grain yield-related traits.

[0155] Test Example 3

[0156] (1) Tannin can reduce ammonium ferric citrate in an alkaline solution to form a dark blue compound. The color depth is proportional to the tannin content. The tannin content is calculated by measuring the absorbance value at a specific wavelength using a standard curve. The target compound was quantitatively analyzed using a Thermo Fisher Scientific (USA) multifunctional microplate reader, model Multiskan GO. The microplate was made from Corning (USA), and the inter-well difference was required to be less than 0.002 at the target wavelength. Determination of total tannin content in sorghum grains:

[0157] 1) Sample Pretreatment: Accurately weigh 0.1 g of sorghum kernel sample into a 15 mL test tube. Add 1 mL of dimethylformamide (DMF) solution and seal the tube. Place the tube in an oscillator and extract for 60 minutes. Centrifuge at 4000 rpm for 10 minutes and separate the supernatant to obtain the extract.

[0158] Among them, SbMyb1-KO was obtained by mixing SbMyb1-KO-1 grains and SbMyb1-KO-2 grains of equal mass.

[0159] 2) Absorbance determination of the first batch of test tubes: Add 0.25 mL of the extract to the first batch of test tubes; add 1.5 mL of deionized water and 0.25 mL of ammonia solution to the first batch of test tubes; shake to mix, and let stand at room temperature for 10 minutes; use a spectrophotometer to measure the absorbance value A1 at a wavelength of 525 nm.

[0160] 3) Absorbance measurement of the second batch of test tubes: Add 0.25 mL of the extract to the second batch of test tubes. Then, add 1.25 mL of deionized water, 0.25 mL of ammonia solution, and 0.25 mL of ammonium ferric citrate solution to the second batch of test tubes. Vortex to mix thoroughly and let stand for 10 minutes. Measure the absorbance (A2) at 525 nm using a spectrophotometer.

[0161] 4) Calculation of sample tannin absorbance (A):

[0162] A=A2-A1;

[0163] Among them, A is the absorbance of tannin in the sample, A1 is the absorbance value of the first batch of test tubes, and A2 is the absorbance value of the second batch of test tubes.

[0164] 4) Quantification was performed using an external standard method. Fitting curves were prepared using different concentrations of standard (0.01, 0.1, 0.2, 0.3, 0.4, 0.5, 1, and 2 mg / mL tannic acid). Tannin content was calculated based on the fitting curves.

[0165] Tannin content (mg / g) = C1 × V1 ÷ M1;

[0166] Wherein, C1 is the tannin concentration calculated by substituting the absorbance measured in step 3) into the fitting curve; M1 is the actual weighed grain mass; V1 is the total volume of the extract.

[0167] (2) Anthocyanin is a water-soluble pigment that changes color with the acidity and alkalinity of the cell fluid. After the sample is extracted with weak acid, the supernatant is taken. Anthocyanin appears red under acidic conditions and has a characteristic absorption peak at around 530nm. The size of the absorption peak is directly proportional to the sample content. The target substance was quantitatively analyzed using a Thermo Fisher (USA) multifunctional microplate reader, the instrument model is Multiskan GO. The microplate was made by Corning (USA), and the difference between wells was required to be less than 0.002 at the target wavelength. Determination of total anthocyanin content in sorghum grains:

[0168] 1) Sample Pretreatment: Accurately weigh 0.1 g of sorghum kernel sample and add 1 mL of 5 (v / v)% formic acid aqueous solution. Vortex mix thoroughly and perform ultrasonic extraction (30°C, 15 min / 10 mL, frequency 40 kHz). Centrifuge the ultrasonic extract at 12,000 rpm and 4°C for 10 min, collect the supernatant into a new centrifuge tube, and continue extraction by adding 1 mL of 5 (v / v)% formic acid aqueous solution to the precipitate. Repeat this step several times until the supernatant is colorless. Combine all supernatants and measure absorbance at 530 nm, using 5% (v / v) formic acid aqueous solution as a blank.

[0169] Among them, SbMyb1-OE was obtained by mixing SbMyb1-OE-1 grains and SbMyb1-OE-2 grains of equal mass.

[0170] 2) Calibration of the standard curve: Quantification was performed using the external standard method. A fitting curve was prepared using different concentrations of standard solution (0.1, 0.2, 0.5, 0.8, 1, 2, 4, 6, 8, 10, and 20 μg / mL cyanidin-3-oxo-sophoroside standard solutions). Anthocyanin content was calculated based on the fitting curve.

[0171] Anthocyanin content (μg / g) = C2 × V2 ÷ M2;

[0172] Wherein, C2 is the anthocyanin concentration calculated by substituting the absorbance measured in step 3) into the fitting curve; M2 is the actual weighed grain mass; V2 is the total volume of the supernatant.

[0173] Test results such as Figure 7 shown.

[0174] In the SbMyb1 gene-edited mutant line (P184), both tannin and anthocyanin contents were significantly reduced. In the SbMyb1 gene-overexpressing line (P226), anthocyanin content was significantly increased. These results suggest that the SbMyb1 gene positively regulates the synthesis of tannins and anthocyanins in grains.

[0175] The embodiments of the present invention are described in detail above in conjunction with the embodiments, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge scope of ordinary technicians in the relevant technical field without departing from the purpose of the present invention.

Claims

1. Use of the SbMyb1 gene or its related biological materials in any one of A1) to A9): A1) Regulate plant grain length; A2) regulating plant grain width; A3) regulating plant thousand-grain weight; A4) regulating the number of grains per ear of plants; A5) Regulates the cell number of endosperm cells in plant grains; A6) regulating the total cell area of endosperm cells in plant grains; A7) regulating tannin synthesis in plants; A8) Regulates anthocyanin synthesis in plants; A9) Plant breeding; The SbMyb1 gene encodes any one of the following proteins B1) to B3): B1) a protein with an amino acid sequence as shown in SEQ ID NO: 2; B2) a protein obtained by substituting and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID NO: 2; B3) A fusion protein obtained by linking a tag to the N-terminus and / or C-terminus of the protein shown in B1) or B2).

2. The use according to claim 1, characterized in that The amino acid sequence of the protein in B2) is shown in SEQ ID NO: 17, SEQ ID NO: 19 or SEQ ID NO:

21.

3. The use according to claim 1, characterized in that The biomaterial comprises at least one of C1) to C7): C1) a nucleic acid molecule encoding the protein; C2) a nucleic acid molecule that increases the expression of the gene encoding the protein or the activity or content of the protein; C3) a nucleic acid molecule that inhibits the expression of the gene encoding the protein or the activity or content of the protein; C4) an expression cassette containing the nucleic acid molecule according to any one of C1) to C3); C5) a recombinant vector containing the nucleic acid molecule of any one of C1) to C3) or the expression cassette of C4); C6) a recombinant microorganism containing the nucleic acid molecule of any one of C1) to C3), the expression cassette of C4), or the recombinant vector of C5); C7) A transgenic cell containing the nucleic acid molecule of any one of C1) to C3), the expression cassette of C4), or the recombinant vector of C5).

4. The use according to claim 3, characterized in that The nucleotide sequence of the nucleic acid molecule in C1) is as shown in any one of C11) to C13): C11) a DNA molecule with a nucleotide sequence as shown in SEQ ID NO: 3, SEQ ID NO: 18, SEQ ID NO: 20 or SEQ ID NO: 22; C12) a DNA molecule that has 80% or more identity with the nucleotide sequence shown in SEQ ID NO: 3, SEQ ID NO: 18, SEQ ID NO: 20, or SEQ ID NO: 22 and encodes a protein having the same function; C13) A DNA molecule that hybridizes with the DNA molecule described in C11) or C12) under stringent conditions and encodes a protein having the same function.

5. The use according to claim 1, characterized in that The plant breeding described in A9) includes any one of A91) to A98): A91) Cultivating plants with increased or decreased grain length; A92) Cultivating plants with increased or decreased grain width; A93) Cultivating plants with increased or decreased 1000-grain weight; A94) Breeding plants with increased or decreased number of grains per ear; A95) Cultivating plants in which the cell number of endosperm cells in grains is increased or decreased; A96) Cultivating plants in which the total cell area of endosperm cells in grains is increased or decreased; A97) Breeding plants with increased or decreased tannin content; A98) Breeding plants with increased or decreased anthocyanin content.

6. A method for cultivating plants with increased or decreased grain length, grain width, 1000-grain weight, number of grains per ear, number of cells in grain endosperm cells, total cell area of grain endosperm cells, tannins and / or anthocyanins, characterized in that: The method comprises: Increasing or inhibiting the expression level of the gene encoding the protein of claim 1 or the activity or content of the protein in the target plant.

7. The method according to claim 6, characterized in that The increasing of the expression level of the gene encoding the protein in the target plant is achieved by introducing a nucleic acid molecule encoding the protein into the target plant.

8. The method according to claim 6, characterized in that The inhibition of the expression level of the gene encoding the protein in the target plant is achieved by knocking out or mutating the gene encoding the protein in the target rice plant.

9. The method according to claim 8, characterized in that The inhibition of the expression of the protein encoding gene in the target plant is achieved by CRISPR / Cas9 technology.

10. The method according to claim 9, characterized in that The target sequences in the CRISPR / Cas9 technology are shown in SEQ ID NO:8 and SEQ ID NO:9.

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