Use of ZmMIE2 protein in regulating quality traits of corn kernels

Abstract

This invention belongs to the field of molecular biology and relates to the application of ZmMIE2 protein in regulating maize kernel quality traits. The amino acid sequence of the ZmMIE2 protein is shown in SEQ ID NO.2, and the nucleotide sequence of the gene encoding the ZmMIE2 protein is shown in SEQ ID NO.1. This invention discovers that the ZmMIE2 protein can regulate maize kernel quality traits, thereby reducing the concentration of certain nutrients in maize kernels. ZmMIE2 Gene overexpression leads to larger kernels, increased 100-kernel weight, and increased soluble sugar content, which can be used to increase corn yield. ZmMIE2 Gene mutations can increase the protein content of kernels, and this invention lays the theoretical foundation for creating new high-protein maize varieties. This discovery not only provides a scientific basis for understanding the development mechanism of kernels, but also provides theoretical guidance for the genetic improvement of maize.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology, specifically the application of ZmMIE2 protein in regulating maize kernel quality traits. Background Technology

[0002] Imprinted genes are a special phenomenon of gene expression regulation, referring to genes in which alleles from the paternal and maternal parents exhibit differential expression in offspring; that is, only one parent's allele is expressed, while the other is not expressed or has very low expression levels. Imprinted genes can be divided into two categories: those genes from the maternal parent whose expression is higher than that from the paternal parent's are defined as maternally expressed genes (MEGs), and those from the paternal parent whose expression is lower are defined as paternally expressed genes (PEGs). If a gene is maternally expressed, only the maternal allele will be expressed in the offspring; conversely, if a gene is paternally expressed, only the paternal allele will be expressed in the offspring. Imprinted genes were first discovered in the endosperm of maize. In 1970, Kermicle discovered the first imprinted gene in the endosperm of maize. R1 This gene primarily regulates anthocyanin synthesis in maize kernels, and its expression in the endosperm has a maternal effect. The inheritance pattern of imprinted genes does not conform to Mendelian inheritance; it is an epigenetic phenomenon. The main factors regulating gene imprinting include DNA methylation, PRC2 complex regulation of histone modifications, non-coding RNA, and different transposon elements. In different species, imprinted genes are located near different transposon elements, and the movement or activation of these different transposon elements affects imprinted gene expression to varying degrees. Subsequent research has demonstrated that imprinted genes participate in regulating many processes of kernel development, such as morphogenesis, dormancy, and postzygotic reproductive isolation. Hundreds of imprinted genes have been identified in different plants using high-throughput techniques, but only a few have had their functions studied in detail. Therefore, exploring the causes and regulatory mechanisms of imprinted genes is helpful in understanding the developmental mechanisms of maize kernels, thus providing a theoretical basis for improving maize yield and quality.

[0003] RNA-binding proteins (RBPs) are a class of proteins that specifically bind to RNA molecules. They participate in various stages of RNA metabolism within cells, including transcription, processing, transport, localization, translation, and degradation. In plants, the RNA-binding protein family includes serine-arginine-rich RNA-binding proteins (SR proteins), glycine-rich RNA-binding proteins (GR-RBPs), trigonal pentapeptide repeat proteins (PPR proteins), DEAD-box RNA helicases (DEAD-box RHs), and RNA chaperones. RNA-binding proteins contain RNA recognition motifs (RRMs), zinc finger motifs, K-homology domains (KH), and cold shock domains. These conserved motifs and domains combine to form various specific RBPs. In eukaryotes, RNA-binding proteins are important post-transcriptional regulators. They can bind to RNA to form ribonucleoprotein complexes, thereby regulating various RNA metabolic processes in eukaryotic cells, such as RNA transfer, modification, translation, and RNA degradation. In addition to participating in RNA metabolism, RNA-binding proteins can also participate in plant growth and development as well as stress responses.

[0004] The RNA Recognition Motif (RRM) domain, also known as the RNA recognition motif, is one of the most common RNA-binding domains. It consists of approximately 90 amino acids and contains two highly conserved sequence motifs: RNP1 (Ribonucleoproteins 1) and RNP2, containing 8 and 6 conserved amino acids respectively. The amino acid residues in these motifs participate in RNA interaction. The RRM domain has a typical β-α-β-α-β secondary structure, where the β-sheet forms a platform for RNA binding, while the α-helix acts as a stabilizing agent. RNP1 and RNP2 are flanked by hydrophobic amino acids arranged irregularly and randomly. The β-α-β-α-β secondary structure of the RRM domain forms four antiparallel β-sheet structures, with the two α-helices perpendicular to the direction of the β-sheet structures. RNP1 and RNP2 are located in the middle of the β3 and β1 sheet structures, respectively. By observing the three-dimensional structure of the RRM, we can see that the RRM structure has two spatial structures with different functions. In the first structure, the charged and aromatic side chains of RNP1 and RNP2 are exposed on the outside, and the outer structure may bind directly to RNA through hydrogen bonds and loop stacking forces. In the second structure, the aromatic side chain at the end of RNP1 folds inward, and other highly conserved hydrophobic amino acids of these two α-helices form the hydrophobic core of the RRM domain. Many RRM domain proteins contain multiple RRM domains, which can be arranged in tandem and work synergistically to improve the binding specificity and affinity for RNA. Previous studies have shown that RNA-binding proteins with RRM domains participate in the splicing of precursor mRNA, affect mRNA polyadenylation, and contribute to mRNA degradation and stability.

[0005] In summary, RNA-binding proteins of the RRM domain can affect grain development through multiple pathways. Therefore, research on RNA-binding proteins of the RRM domain will help us further understand the regulatory mechanisms of grain development. Summary of the Invention

[0006] Based on the above-mentioned technical problems, the present invention provides the application of ZmMIE2 protein in regulating maize kernel quality traits.

[0007] The first objective of this invention is to provide the application of the ZmMIE2 protein or its encoding gene in regulating maize kernel quality traits, wherein the amino acid sequence of the ZmMIE2 protein is shown in SEQ ID NO.2, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.1.

[0008] The inventors previously discovered, through screening imprinted gene mutants in corn kernels, that maternal imprinted genes...ZmMIE2 Gene mutations may affect kernel morphology and development. However, whether the ZmMIE2 protein regulates kernel area, embryo area, and the content of soluble glycoproteins and proteins in maize kernels is unknown. This invention... ZmMIE2 Gene overexpression and gene mutation have demonstrated that promoting the expression of the ZmMIE2 protein can increase the kernel area, 100-kernel weight, yield, and soluble sugar content of maize, while inhibiting the expression of the ZmMIE2 protein can increase the protein content of maize kernels. This invention provides a precisely controllable single-gene strategy and a novel breeding target for the synergistic improvement of high-yield and high-quality maize.

[0009] Furthermore, the expression of the ZmMIE2 protein is promoted by transferring biological material containing the encoding gene into maize.

[0010] Furthermore, the biological material is a recombinant vector, expression cassette, transgenic cell line, or recombinant bacteria.

[0011] Furthermore, the inhibition of the expression of the ZmMIE2 protein was achieved through gene editing using the CRISPR / Cas9 system.

[0012] Furthermore, the CRISPR / Cas9 system is a recombinant vector expressing gRNA and Cas9.

[0013] Furthermore, the target sequence of the gRNA is shown in SEQ ID NO.5.

[0014] A second objective of this invention is to provide a method for cultivating transgenic maize with increased kernel area, higher 100-kernel weight, and increased soluble sugar content, including a step of promoting the expression of the ZmMIE2 protein in maize.

[0015] A third objective of this invention is to provide a method for breeding transgenic maize with increased grain protein content, including the step of inhibiting the expression of the ZmMIE2 protein in maize.

[0016] The present invention has the following beneficial effects:

[0017] This invention is the first to discover in corn ZmMIE2 Gene overexpression can increase the kernel area, 100-kernel weight, and soluble sugar content of maize kernels, while decreasing the protein content. This discovery not only provides a scientific basis for understanding the development mechanism of kernels but also offers theoretical guidance for the genetic improvement of maize. Attached Figure Description

[0018] Figure 1 for ZmMIE2 Identification of gene structure and frameshift mutant lines; A: ZmMIE2B: Target sites for gene editing materials from gene overexpression lines; ZmMIE2 Gene mutation sites and types.

[0019] Figure 2 To obtain by quantitative fluorescence experiment ZmMIE2 Gene expression profile.

[0020] Figure 3 for ZmMIE2 Comparison of grain area and length / width between self-pollinated gene-edited lines, overexpression lines, and wild-type lines; A: Quantitative comparison of grain area, B: Quantitative comparison of grain width, C: Quantitative comparison of grain length, D: Typical images of grain width (left) and length (right). **: Indicates extremely significant at P<0.01.

[0021] Figure 4 This is a schematic diagram of the ZmMIE2 protein structure. WT: Wild-type KN5585; ZmMIE2 -C1, ZmMIE2 -C2: ZmMIE2 Two frameshift mutant lines.

[0022] Figure 5 This is a schematic diagram of the construction of the GFP recombinant plasmid.

[0023] Figure 6 Full-length cells in tobacco leaf epidermal cells ZmMIE2 Subcellular localization of the GFP fusion protein.

[0024] Figure 7 for ZmMIE2 Comparison of 100-grain weight, soluble sugar content, starch content, and protein content of self-pollinated seeds from gene-edited lines, overexpression lines, and wild-type lines; A: 100-grain weight, B: soluble sugar content, C: starch content, D: protein content. **: indicates highly significant at P<0.01. Detailed Implementation

[0025] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments are commercially available unless otherwise specified.

[0026] Example 1: Imprinted Genes ZmMIE2 Cloning

[0027] ZmMIE2 The nucleotide sequence of the gene is shown in SEQ ID NO.1. ZmMIE2 The gene encodes the protein ZmMIE2. The amino acid sequence of protein ZmMIE2 is shown in SEQ ID NO.2.

[0028]

[0029] SEQ ID NO.2: MAAAPAASSSTSGSPAAGGPRSRFGDTTLTKVFVGGLAWETPSEGLRQHFERYGDILEAVVITDRLTGRSKGYGFVTFREPEAARRAVQDPNPTIAGRRANCNIASLGPPRPTQPGVAGRGGPYTVGPHHLQVPQFVPRAPASPLQMMVPQQQQQHGGAPAAAI YPSPQFGYCWYPPDFQYQQALASPQALQNYYAQLYGLTTSPSAAAAPYHHQYLGYMAPPPPTPRMILPPPPPLAAQQVTAVQPLVQHPPPPAQQVTVQPLLQHPPPQIHAPFFPAPSLPQHNFRLHPPPQAMAVLPPNTTAGGSLPPADQAAAPAARATNASSTRPGA.

[0030] Example 2, Gene ZmMIE2 Overexpression and construction of gene-edited lines

[0031] 1. Genes ZmMIE2 Construction of overexpression lines:

[0032] 1) Maize genome annotation database MaizeGDB was retrieved. ZmMIE2 The complete nucleotide sequence of the gene, as shown in SEQ ID NO.1, was extracted from maize variety KN5585. ZmMIE2 RNA from specifically expressed tissues was extracted and reverse transcribed to obtain cDNA. This cDNA was then amplified by PCR and purified using ZmMIE2-CDS-F (SEQ ID NO.3) and ZmMIE2-CDS-R (SEQ ID NO.4) to obtain... ZmMIE2 The CDS sequence was obtained, and the PCR purified product was sent to Shanghai Sangon Biotech Co., Ltd. for sequencing.

[0033] SEQ ID NO.3: 5'-CTCCCTGCTCGCAAGACCA-3';

[0034] SEQ ID NO. 4: 5'-AGTTCTCCTTGAAGCCTCTCTG-3'.

[0035] 2) Amplification was performed using a high-fidelity enzyme (Yeasen). After gel electrophoresis, PCR products with correctly identified bands were recovered. The pCambia1300 vector was linearized using restriction enzymes KpnI and XbaI. ZmMIE2 Recombination of the CDS region of a gene with a linearized vector, such as Figure 1 As shown in Figure A, the recombinant vector was transformed into Escherichia coli DH5α strain.

[0036] 3) After the selected single colonies are tested, they are sent to the company for sequencing.

[0037] 4) Take the strain with correct sequencing results, extract the plasmid according to the steps shown in the plasmid rapid extraction kit (TIANGEN), and store it in a -20℃ refrigerator for later use.

[0038] 2. Genes ZmMIE2 Construction of gene editing vectors:

[0039] (1) First choose to be close to ZmMIE2 A 19bp target sequence (shown in SEQ ID NO.5) was designed for the CDS region of the gene translation start codon, and primers for the target sequence were synthesized (shown in SEQ ID NO.6 and SEQ ID NO.7).

[0040] SEQ ID NO.5: 5′-ACTGCAACATTGCGTCGCT-3′ (i.e., bits 715-733 of SEQ ID NO.1 from the end of 5′).

[0041] SEQ ID NO.6: 5'-TTTGTGCATTTACTTGGGCTA-3';

[0042] SEQ ID NO. 7: 5'-TGAGCACCCCATTGCCTA-3'.

[0043] (2) The intermediate vector pCBC-MT1T2 (published in "Xing HL, Dong L, Wang ZP, Zhang HY, Han CY, Liu B, Wang XC, Chen QJ. A CRISPR / Cas9 toolkit for multiplex genomeediting in plants. BMC Plant Biol. 2014 Nov 29;14:327.") was used as a template for amplification, and the PCR product containing the target sequence was purified and recovered;

[0044] (3) Using 10×BsaI restriction enzyme and high concentration of T4 ligase, the PCR product and pBUE411 vector were ligated, transformed into E. coli and single clones were selected for identification. The correctly ligated single clones were selected, shaken and the plasmid was extracted and stored at -20℃.

[0045] 3. Agrobacterium competent cell transformation and Agrobacterium infection of transgenic organisms

[0046] Refer to Chinese patent number CN202410959611.2.

[0047] Example 3, Imprinted Genes ZmMIE2 Grain phenotype identification of gene-edited mutant lines and overexpression lines

[0048] 0. Imprinted genes ZmMIE2 Genotyping of overexpression lines

[0049] 1. Using genomic DNA from T0 generation transgenic maize leaves as templates, the specific Bar gene sequence on the transgenic vector was amplified (using SEQ ID NO. 8 and SEQ ID NO. 9) to screen for overexpression-positive plants. PCR amplification was performed using primers consisting of SEQ ID NO. 10 and SEQ ID NO. 11 (procedure shown in Table 1), yielding the corresponding PCR amplification products. The PCR amplification products were then subjected to Sanger sequencing. Sequencing results were compared with... ZmMIE2 The target site sequences edited by the CAS9 protein in the genes were compared to select plants with heterozygous or homozygous frameshift mutations in the target site region.

[0050] SEQ ID NO. 8: 5'-GCAAAGTCTGCCGCCTTACAAC-3'.

[0051] SEQ ID NO.9: 5'-TGTTATCCGCTCACAATTCCACAC-3'.

[0052] SEQ ID NO. 10: 5'-GGCAACACTTCGAGCGGTA-3'.

[0053] SEQ ID NO. 11: 5'-GACTTTTCTCAGCTTCAGCCAG-3'.

[0054] Table 1 PCR reaction procedure

[0055]

[0056] II. Imprinted Genes ZmMIE2 Genotyping of gene-edited strains

[0057] 1. Self-pollinate the heterozygous mutants identified above to obtain seeds, which are T1 generation seeds, and plants grown from T1 generation seeds are T1 generation plants; self-pollinate the T1 generation plants to obtain seeds, which are T2 generation seeds, and plants grown from T2 generation seeds are T2 generation plants.

[0058] 2. Using genomic DNA from leaves of the T1 generation plants as templates, PCR amplification was performed using primer pairs consisting of SEQ ID NO. 6 and SEQ ID NO. 7 to obtain the corresponding PCR amplification products. The PCR amplification products were then subjected to Sanger sequencing. The sequencing results were compared with... ZmMIE2 The target sequences of genes edited by the CAS9 protein are compared to select homozygous mutation types.

[0059] 3. ZmMIE2- The two homologous chromosomes of C1 ZmMIE2 The same mutation occurred in the genes, specifically on two homologous chromosomes. ZmMIE2 The genes all have a single T base insertion (730bp-731bp). Figure 1 B), which causes a frameshift, resulting in the loss of function of the protein ZmMIE2.

[0060] 4. ZmMIE2- The ZmMIE2 gene on both homologous chromosomes of C2 underwent the same mutation, specifically on both homologous chromosomes. ZmMIE2 The genes all have an insertion of one A base (730bp-731bp). Figure 1 B), which causes a frameshift, resulting in the loss of function of the protein ZmMIE2.

[0061] 5. Combination ZmMIE2 Gene expression profile data in maize ( Figure 2 The gene was found to be highly expressed in grains, so it was targeted... ZmMIE2 The grain area, grain width, and grain length of the gene mutants and overexpression lines were measured. The results showed that the two lines... ZmMIE2 The seed area of ​​gene-edited lines was significantly smaller than that of wild type, while the seed area of ​​overexpression lines was significantly larger than that of wild type. Figure 3 Further research revealed that the core factor affecting grain area is a significant change in grain length.

[0062] Example 3 ZmMIE2 Results of gene protein structure and subcellular localization

[0063] 1. For each ZmMIE2 In gene-edited mutants and overexpression lines, ZmMIE2 Protein structure prediction of related gene sequences revealed that the protein structures of both gene-edited mutants were significantly altered compared to the wild type, which may be a key factor ultimately affecting grain phenotype. Figure 4 ).

[0064] 2. To further investigate the localization of this gene in cells, we conducted... ZmMIE2Subcellular localization experiments of genes. Genes with correct sequencing... ZmMIE2 The purified CDS sequence product was ligated into the T-vector (GENSTAR, T184). Figure 5 The reaction system is as follows:

[0065] Table 2 Reaction systems for T-carrier linkage

[0066]

[0067] The prepared system was reacted at 37°C for 5 minutes.

[0068] 3. The ligated product was directly transformed into competent E. coli DH5α cells. The transformation process is as follows: 1) The extracted competent E. coli DH5α cells were thawed on ice;

[0069] 2) Add an appropriate amount of ligation reaction solution to competent cells (volume of ligation reaction solution ≤ 10% of competent cell volume), mix gently, and incubate on ice for 30 minutes;

[0070] 3) Incubate in a 42°C water bath for 2 minutes, then immediately place in ice for 2 minutes;

[0071] 4) Add 500 μl of SOC medium and place in a shaker at 37°C, 180 rpm, for 1 h to activate E. coli;

[0072] 5) Spread the bacterial suspension evenly onto LB agar plates containing 50 μg / ml kanamycin and incubate at 37°C for 12-16 h.

[0073] 4. Pick a single clone and add it to a 2.0 ml centrifuge tube. Add 1.5 ml of LB medium (containing 50 μg / ml kanamycin) to the centrifuge tube and incubate at 37℃ and 180 rpm for 3-4 h in a shaker. Perform PCR on the bacterial culture using M13 primers (using SEQ ID NO.12 and SEQ ID NO.13). Send the bacterial culture to Sangon Biotech (Shanghai) Co., Ltd. for sequencing identification. Extract the plasmid with the correct sequence and store it at -20℃ for later use.

[0074] SEQ ID NO.12: 5'-TGTAAAACGACGGCCAGT-3';

[0075] SEQ ID NO. 13: 5'-CAGGAAACAGCTATGACC-3'.

[0076] 5. Enzyme digestion: The empty GFP vector was digested using two restriction enzymes, KpnI and XbaI.

[0077] Table 3. Enzyme digestion systems of KpnI and XbaI endonucleases

[0078]

[0079] The reaction procedure was as follows: 37℃, 1h, 75℃, 15min, followed by gel recovery using an agarose gel DNA recovery kit (TIANGEN, DP210) to obtain the GFP linearized vector.

[0080] 6. Using primers (ZmMIE2-GFP) containing homologous recombination-specific sequences, the extracted... ZmMIE2 Using the plasmid as a template, PCR amplification was followed by gel recovery to obtain plasmids with added GFP homologous arms. ZmMIE2 The CDS sequence is used for subsequent connections.

[0081] 7. Homologous recombination: Homologous recombination was performed using GENSTAR's EZ-Flex Seamless Cloning Kit (T197). The reaction system is as follows:

[0082] Table 4 Reaction systems for homologous recombination

[0083]

[0084] Mix gently and react at 37°C for 30 min. After the reaction, place on ice and directly transform the product into competent E. coli DH5α cells. After plating, picking, and culturing, PCR identification was performed using a combination of gene-specific primers (SEQ ID NO.14) and GFP vector-specific primers (SEQ ID NO.15). Bacterial solutions with bands in the correct positions were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. Plasmids were extracted from the correctly sequenced bacterial solutions to obtain recombinant GFP plasmids.

[0085] SEQ ID NO.14: 5'-ACGGGGGACGAGCTCGATGGCAGCAGCCCCGGCGG-3';

[0086] SEQ ID NO. 15: 5'-GCTTCATGTGGTCGGGGTAGC-3'.

[0087] 8. Agrobacterium-mediated transformation

[0088] 1) Thaw competent cells of GV3101 Agrobacterium (Video Biotechnology, AC1002) taken out at -80℃ on ice;

[0089] 2) Add 2 μl of the constructed recombinant GFP plasmid to every 50 μl of competent cells in a 1.5 ml centrifuge tube, and incubate on ice for 30 min;

[0090] 3) Quick freeze in liquid nitrogen for 5 minutes, then in a 37°C water bath for 5 minutes; quickly transfer to ice and let stand for 5 minutes;

[0091] 4) Add 500 μl of SOC medium to the centrifuge tube, incubate at 28℃ and 200 rpm for 2-3 hours to recover;

[0092] 5) Take 200 μl of bacterial suspension and spread it on a solid culture medium containing kanamycin antibiotic;

[0093] 6) Invert the container and incubate at 28°C in the dark for 48-72 hours;

[0094] 7) Prepare a 2.0ml sterile centrifuge tube, add 1.5ml of liquid culture medium containing kanamycin and rifampin, pick each single colony and place it in a new centrifuge tube, incubate at 28℃ and 200rpm until the bacterial solution becomes turbid (about 12h).

[0095] 8) Using the gene-specific primers (SEQ ID NO.12) and the GFP vector-specific primers (SEQ ID NO.13), and with the turbid bacterial solution as a template, perform bacterial testing to confirm successful plasmid transformation;

[0096] 9) Take 700 μl of the successfully transformed bacterial culture, add 300 μl of glycerol, mix by pipetting, and store at -80℃ for long-term storage.

[0097] 9. Instantaneous conversion of tobacco leaves

[0098] 1) Prepare a 50ml sterile centrifuge tube, add 25ml of LB medium containing kanamycin and rifampin, add 100μl of Agrobacterium tumefaciens containing the plasmid to be transformed, and incubate at 28℃ and 200rpm for more than 12 hours.

[0099] 2) The concentration of the bacterial culture was determined using a spectrophotometer, with an OD600 value between 0.6 and 1.0;

[0100] 3) Centrifuge at 3500g for 15 minutes, discard the supernatant, and repeat once to remove antibiotics as much as possible;

[0101] 4) Finally, adjust the bacterial suspension to OD600≈1 using the resuspension solution;

[0102] 5) At the same time, Agrobacterium containing the P19 plasmid was also suspended and the concentration of the bacterial solution was adjusted to OD600≈1;

[0103] 6) Based on the final amount of bacterial solution used (3 mL is needed to completely wet each leaf), in a new centrifuge tube, mix the bacterial solution containing the target plasmid (or marker plasmid) with the bacterial solution containing the P19 plasmid in an equal volume ratio of 1:1, and place the centrifuge tube at 28°C and incubate in the dark for 2-5 hours.

[0104] 7) Select healthy tobacco leaves (fully unfolded leaves on plants with about 6-7 leaf age) for Agrobacterium infection. Water thoroughly 2-3 days before transformation and control watering after transformation.

[0105] 8) Before inoculation, place the tobacco under a white fluorescent light for 1 hour to allow the pores to open;

[0106] 9) Before injection, resuspend the bacterial cells and draw up the bacterial solution with a 1ml syringe;

[0107] 10) When injecting the bacterial solution, first make a small hole on the back of the leaf with the needle of the syringe. Hold the front of the leaf with your left hand and gently press the syringe tube (without the needle) vertically onto the back of the leaf with your right hand. Slowly push with your right thumb and observe the bacterial solution moving slowly under the leaf epidermis until it infects the entire leaf. Label the injected petiole (2-3 leaves can be injected per plant, do not use cotyledons. If the leaves are large, 2-4 small holes can be made in different locations).

[0108] 11) Keep the tobacco moist after injection and continue culturing for 2-5 days;

[0109] 12) Before observation, prepare several clean glass slides, drop a drop of ddH2O in the center of the slide, tear off the lower epidermis of the tobacco leaf and place it on the glass slide, and gently cover it with a coverslip from one side to avoid air bubbles.

[0110] 13) Fluorescence signals were observed under a laser confocal microscope, and co-localization of the signal points with chloroplast markers was found, confirming that the ZmMIE2 protein is located on chloroplasts. Figure 6 ).

[0111] Example 4 ZmMIE2 Elemental analysis of grains from homozygous mutant lines and overexpression lines

[0112] 1. Material planting (planting) ZmMIE2 overexpression plants, ZmMIE2 Frameshift mutant lines and wild-type KN5585). ZmMIE2 Overexpression and wild-type self-pollination, ZmMIE2 Frameshift mutant lines ZmMIE2 Frameshift mutant lines with different transformation events were crossed orthogonally and reciprocally with wild-type lines, and three plants were taken from each pollination method. The weight of 100 grains, protein, starch and soluble sugar content were measured at 15 days after pollination and at maturity.

[0113] a. Determination of soluble sugars

[0114] Preparation of stock solution: Dissolve 100 mg of glucose, which has been dried to constant weight in an 80°C oven, in 80% (v / v) ethanol to prepare a 1000 mL solution to obtain the glucose standard solution. Weigh 1 g of anthrone and dissolve it in 1000 mL of dilute sulfuric acid solution to obtain the anthrone reagent, which is stored in a brown bottle and prepared for use on the same day.

[0115] 1) Sample extraction

[0116] Place the test seeds in an oven at 110℃ for 15 minutes, then adjust the temperature to 70℃ and leave overnight. After the seeds are completely dry, grind them and weigh 0.05g. Place the powder in a 10ml centrifuge tube, add 4ml of 80% ethanol, and place the sample in an 80℃ water bath for 40 minutes, stirring every 10 minutes. Then centrifuge at 5000g for 5 minutes and collect the supernatant. Add 0.01g of activated carbon to the supernatant and decolorize at 80℃ for 30 minutes. Finally, bring the volume to 10ml and use the filtrate for analysis.

[0117] 2) Draw the standard curve

[0118] Prepare glucose solutions of different concentrations, add 5 mL of freshly prepared anthrone reagent, mix well, and boil in a water bath for 10 min, then quickly transfer to cold water to cool for 2 min. Measure the absorbance at 625 nm using a spectrophotometer. Plot a standard curve with glucose concentration on the x-axis and absorbance on the y-axis, and calculate the standard linear equation.

[0119] 3) Measurement

[0120] Take 1 ml of the filtrate from step 1), mix it with 5 ml of anthrone reagent, measure the absorbance using the same method as in step 2), and determine the soluble sugar content according to the standard curve.

[0121] b. Starch determination

[0122] Take 0.5 g of the residue from step (1) of ethanol extraction of soluble sugars, place it in an Erlenmeyer flask, add 25 ml of 20 g / L HCl, boil in a water bath for 3.5 h, neutralize with 5 mol / L NaOH, add Ba(OH)2 until complete precipitation, and add one drop of phenolphthalein indicator. Add ZnSO4 while stirring. Precipitate barium salt by adding more solution until the red color fades, and then add more Ba(OH)2 solution until it returns to a light red color as the stopping point. Filter and dilute to 250 ml. Take 2 ml to determine its glucose content.

[0123] Calculate crude starch content = glucose content × 0.9; protein determination.

[0124] c. Protein determination

[0125] Using a Starlink G3100 near-infrared grain analyzer to analyze grains ZmMIE2 Overexpression of self-pollinated seeds ZmMIE2 The frameshift mutation self-pollinated grains and wild-type self-pollinated grains were tested. During the test, each bag of material should be filled to the test column. The bottom of the test column should be tapped to ensure that the test material fills the test column completely without leaving any gaps. The test was performed three times, and the data were processed and analyzed using Excel software.

[0126] d. Determination of 100-grain weight

[0127] After air drying ZmMIE2 Self-pollinated fruit ears of overexpression lines ZmMIE2 After threshing, 100 kernels were taken from each frameshift mutant self-pollinated ear and wild-type self-pollinated ear and their 100-kernel weight was measured. Each ear was weighed 3 times, and 3 ears of each material were taken to calculate the 100-kernel weight.

[0128] 2. ZmMIE2 The results of the comparison of internal elemental analysis of grains between the mutant and the wild-type overexpression line are as follows: Figure 7 As shown, ZmMIE2 The mutant seeds showed significantly lower protein content and significantly higher soluble sugar content compared to the wild type. Combined with results from the overexpression lines, as well as two sets of results regarding starch content and 100-seed weight, we can finally draw the following conclusion: ZmMIE2 Genes can influence the development of corn kernels, and overexpression can... ZmMIE2 Genetic modification can increase kernel area and 100-kernel weight, thus increasing corn yield. ZmMIE2 Gene mutations can increase the protein content of kernels, laying a theoretical foundation for the creation of new high-protein corn varieties.

[0129] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

Claims

1. The application of ZmMIE2 protein or its encoding gene in regulating maize kernel quality traits, characterized in that, The amino acid sequence of the ZmMIE2 protein is shown in SEQ ID NO.2, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.

1. Promoting the expression of the ZmMIE2 protein can increase the kernel area, 100-kernel weight, and soluble sugar content of corn kernels, while inhibiting the expression of the ZmMIE2 protein can increase the protein content of corn kernels.

2. The application of the ZmMIE2 protein or its encoding gene according to claim 1 in regulating maize kernel quality traits, characterized in that, The expression of the ZmMIE2 protein is promoted by transferring biological material containing the encoding gene into maize.

3. The application of the ZmMIE2 protein or its encoding gene according to claim 2 in regulating maize kernel quality traits, characterized in that, The biological material is a recombinant vector, expression cassette, transgenic cell line, or recombinant bacteria.

4. The application of the ZmMIE2 protein or its encoding gene according to claim 1 in regulating maize kernel quality traits, characterized in that, Inhibition of the expression of the ZmMIE2 protein was achieved through gene editing using the CRISPR / Cas9 system.

5. The application of the ZmMIE2 protein or its encoding gene according to claim 4 in regulating maize kernel quality traits, characterized in that, The CRISPR / Cas9 system is a recombinant vector expressing gRNA and Cas9.

6. The application of the ZmMIE2 protein or its encoding gene according to claim 5 in regulating maize kernel quality traits, characterized in that, The target sequence of the gRNA is shown in SEQ ID NO.

5.

7. A method for cultivating transgenic corn with increased kernel area, higher 100-kernel weight, and increased soluble sugar content, characterized in that, Includes the step of promoting the expression of the ZmMIE2 protein as described in claim 1 in maize.

8. A method for cultivating transgenic maize with increased grain protein content, characterized in that, Includes the step of inhibiting the expression of the ZmMIE2 protein as described in claim 1 in maize.

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