Application of ZmMIE2 protein in regulating and controlling quality characters of corn kernels

Abstract

The invention belongs to the field of molecular biology, and relates to application of ZmMIE2 protein in regulating and controlling quality characters of corn kernels. The amino acid sequence of the ZmMIE2 protein is as shown in SEQ ID NO.2, and the nucleotide sequence of the ZmMIE2 protein coding gene is as shown in SEQ ID NO.1. The invention further discloses a preparation method of the ZmMIE2 protein. It is found that the ZmMIE2 protein can regulate and control the quality character of corn kernels, overexpression of the ZmMIE2 gene in corn can lead to grain enlargement and increase of hundred-grain weight and soluble sugar content, and the ZmMIE2 protein can be used for improving the corn yield. And the ZmMIE2 gene mutation can improve the protein content of the grains, so that a theoretical foundation is laid for creating a new high-protein corn variety. The discovery not only can provide a scientific basis for analyzing a development mechanism of grains, but also can provide theoretical guidance for genetic improvement of corn.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of molecular biology, and relates to application of ZmMIE2 protein in regulation of corn kernel quality traits. BACKGROUND

[0002] Imprinting is a special gene expression regulation phenomenon, which refers to the gene that exhibits differential expression in offspring from paternal and maternal alleles, i.e., only one allele is expressed, and the other is not expressed or has extremely low expression. Imprinting can be divided into two categories, and the gene from the maternal side of the hybrid offspring is defined as a maternal imprinting gene (MEG) if it is expressed higher than the paternal allele, and vice versa, it is defined as a paternal imprinting gene (PEG). If a gene is a maternal imprinting gene, it is expressed only from the maternal side in offspring, and a paternal imprinting gene is expressed only from the paternal side in offspring. Imprinting was first discovered in the endosperm of corn. In 1970, Kermicle discovered the first imprinting gene in the endosperm of corn R1 , which mainly regulates the synthesis of anthocyanin in corn kernels, and the expression in the endosperm has a maternal effect. The genetic law of imprinting gene does not conform to Mendelian inheritance law, and it is an epigenetic phenomenon. The causes of imprinting gene regulation mainly include DNA methylation, PRC2 complex regulation of histone modification, non-coding RNA and different transposable elements. In different species, different transposable elements are contained near the imprinting gene, and the movement or activation of these different transposable elements will affect the expression of the imprinting gene to different degrees. Continuous research by later generations has proved that imprinting genes can participate in regulating many processes of kernel development, such as morphogenesis, dormancy and postzygotic reproductive isolation. Through high-throughput technology, hundreds of imprinting genes have been identified in different plants, but only a few of them have been studied in detail. Therefore, exploring the causes and regulation mechanism of imprinting genes helps to understand the development mechanism of corn kernels, thereby providing a theoretical basis for improving corn yield and quality.

[0003] RNA binding proteins (RBPs) are a class of proteins that can specifically bind to RNA molecules. They are involved in various aspects of RNA metabolism in 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), pentatricopeptide repeat proteins (PPR proteins), DEAD-box RNA helicases (DEAD-box RHs), and RNA chaperones. RNA binding proteins contain RNA recognition motifs (RRM), 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 that can form ribonucleoprotein complexes by binding to RNA, and then regulate various RNA metabolic processes, such as RNA transfer, modification, translation, and RNA degradation. In addition to participating in RNA metabolism, RNA binding proteins can also be involved in plant growth and development processes and stress responses.

[0004] The RRM (RNA Recognition Motif) domain, also known as the RNA recognition motif, is one of the most common RNA binding domains. It consists of about 90 amino acids, and contains two highly conserved sequence motifs, RNP1 (Ribonucleoproteins 1) and RNP2, which contain 8 and 6 conserved amino acids respectively, and the amino acid residues in the two motifs are involved in the interaction with RNA. The RRM domain has a typical β-α-β-α-β secondary structure, in which the beta sheet forms a platform for binding to RNA, and the alpha helix plays a role in stabilizing the structure. The two sides of RNP1 and RNP2 are composed of some hydrophobic amino acids, which are irregularly scattered, and the β-α-β-α-β secondary structure of the RRM domain forms 4 anti-parallel β sheet structures, and the 2 alpha helices are perpendicular to the direction of the β sheet structure. RNP1 and RNP2 are located at the middle positions of the β3 and β1 sheet structures respectively. By observing the three-dimensional structure of RRM, we can see that the RRM structure has two different spatial structures, the first structure exposes the charged and aromatic side chains of RNP1 and RNP2 on the outside, and the outside structure can directly bind to RNA through hydrogen bonding and ring stacking forces; the second structure folds the aromatic side chain at the last position of RNP1 inward, and the other highly conserved hydrophobic amino acids of the two alpha helices form the hydrophobic core of the RRM domain. Many RRM domain proteins contain multiple RRM domains, which can be arranged in series and work together to improve the binding specificity and affinity of RNA. Previous studies have shown that RNA binding proteins with RRM domains are involved in the splicing of precursor mRNA, affect the polyadenylation of mRNA, and affect the degradation and stability of mRNA.

[0005] In summary, the RNA binding protein with RRM domain can affect the development of grains through various pathways, and therefore the research on the RNA binding protein with RRM domain is helpful for us to further understand the regulation mechanism of grain development. SUMMARY

[0006] Based on the above technical problems, the application provides an application of ZmMIE2 protein in regulating quality traits of corn kernels.

[0007] The first object of the application is to provide an application of ZmMIE2 protein or its coding gene in regulating quality traits of corn kernels, wherein the amino acid sequence of the ZmMIE2 protein is shown as SEQ ID NO. 2. The nucleotide sequence of the coding gene is shown as SEQ ID NO. 1.

[0008] The inventors found that the maternal imprinting gene ZmMIE2 is involved in the regulation of grain development in the early stage of grain development by screening the mutant of imprinting gene in corn kernels.ZmMIE2 The gene mutation can affect the morphological development of the kernel. However, whether the ZmMIE2 protein regulates the kernel area, embryo area, soluble sugar protein content and protein content of the corn kernel is unknown. The present application proves that ZmMIE2 The gene overexpression and gene mutation prove that promoting the expression of the ZmMIE2 protein can increase the kernel area, 100-grain weight, yield and soluble sugar content of the corn kernel, and inhibiting the expression of the ZmMIE2 protein can increase the protein content of the corn kernel, and the present application provides a single gene strategy that can be precisely controlled and a new breeding target for the high-yield and high-quality synergistic improvement of corn.

[0009] Further, the expression of the ZmMIE2 protein is promoted by transferring biological material containing the coding gene into corn.

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

[0011] Further, the expression of the ZmMIE2 protein is inhibited by gene editing through a CRISPR / Cas9 system.

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

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

[0014] The second object of the present application is to provide a method for cultivating transgenic corn with increased kernel area, increased 100-grain weight and increased soluble sugar content, comprising the step of promoting the expression of the ZmMIE2 protein in corn.

[0015] The third object of the present application is to provide a method for cultivating transgenic corn with increased kernel protein content, comprising the step of inhibiting the expression of the ZmMIE2 protein in corn.

[0016] The present application has the following beneficial effects: The present application first discovers that the overexpression of the gene can increase the kernel area of corn, increase the 100-grain weight, increase the soluble sugar content of the corn kernel and reduce the protein content of the corn kernel. ZmMIE2 The present application first discovers that the overexpression of the gene can increase the kernel area of corn, increase the 100-grain weight, increase the soluble sugar content of the corn kernel and reduce the protein content of the corn kernel. The discovery not only provides a scientific basis for analyzing the development mechanism of the kernel, but also provides a theoretical guidance for the genetic improvement of corn. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 To ZmMIE2 Gene structure and identification of frameshift mutant lines; A: ZmMIE2 Target site of gene editing material of gene overexpression line; B:ZmMIE2 Gene mutation sites and types.

[0018] Figure 2 To obtain by quantitative fluorescence experiment ZmMIE2 Gene expression profile.

[0019] 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.

[0020] 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.

[0021] Figure 5 This is a schematic diagram of the construction of the GFP recombinant plasmid.

[0022] Figure 6 Full-length cells in tobacco leaf epidermal cells ZmMIE2 Subcellular localization of the GFP fusion protein.

[0023] 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

[0024] 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.

[0025] Example 1: Imprinted Genes ZmMIE2 Cloning 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.

[0026]

[0027] SEQ ID NO. 2: MAAAP AASSSTSGSPAAGGPRSRFGDTTLTKVFVGGLAWETPSEGLRQHFERYGDILEAVVITDRLTGRSKGYGFVTFREPEAARRAVQDPNPTIAGRRANCNIASLGPPRPTQPGVAGRGGPYTVGPHHLQVPQFVPRAPASPLQMMVPQQQQQHGGAPAAAIYPSPQFGYCWYPPDFQYQQALASPQALQNYYAQLYGLTTSPSAAAAPYHHQYLGYMAPPPPTPRMILPPPPPLAAQQVTAVQPLVQHPPPPAQQVTVQPLLQHPPPQIHAPFFPAPSLPQHNFRLHPPPQAMAVLPPNTTAGGSLPPADQAAAPAARATNASSTRPGA.

[0028] Example 2, Construction of gene ZmMIE2 overexpression strain and gene edited strain 1, Construction of gene ZmMIE2 overexpression strain: 1) The complete nucleotide sequence of ZmMIE2 gene was obtained from the corn genome annotation database MaizeGDB, as shown in SEQ ID NO. 1, and the RNA specifically expressed in the KN5585 variety of corn was extracted and reverse transcribed to obtain cDNA. The CDS sequence of ZmMIE2 was obtained by PCR amplification and purification using ZmMIE2-CDS-F shown in SEQ ID NO. 3 and ZmMIE2-CDS-R shown in SEQ ID NO. 4. ZmMIE2 ZmMIE2 The PCR purification product was sequenced by Shanghai Shengong Biotechnology Co., Ltd. ZmMIE2

[0029] SEQ ID NO. 3: 5'-CTCCCTGCTCGCAAGACCA-3'; SEQ ID NO. 4: 5'-AGTTCTCCTTGAAGCCTCTCTG-3'.

[0030] 2) Amplification was performed using high-fidelity enzyme (Yeasen), and the correct PCR product was recovered by gel electrophoresis detection. The pCambia1300 vector was linearized using restriction enzymes KpnI and XbaI, and the CDS region of the gene was recombined with the linearized vector, as shown in FIG. A. ZmMIE2 Figure 1 The recombinant vector was transformed into E. coli DH5a strain.​​​

[0031] 3) After the selected single colonies are tested, they are sent to the company for sequencing.

[0032] 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.

[0033] 2. Genes ZmMIE2 Construction of gene editing vectors: (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).

[0034] SEQ ID NO.5: 5′-ACTGCAACATTGCGTCGCT-3′ (i.e., bits 715-733 of SEQ ID NO.1 from the end of 5′).

[0035] SEQ ID NO.6: 5'-TTTGTGCATTTACTTGGGCTA-3'; SEQ ID NO. 7: 5'-TGAGCACCCCATTGCCTA-3'.

[0036] (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; (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℃.

[0037] 3. Agrobacterium competent cell transformation and Agrobacterium infection of transgenic organisms Refer to Chinese patent number CN202410959611.2.

[0038] Example 3, Imprinted Genes ZmMIE2 Grain phenotype identification of gene-edited mutant lines and overexpression lines 0. Imprinted genes ZmMIE2 Genotyping of overexpression lines 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.

[0039] SEQ ID NO. 8: 5'-GCAAAGTCTGCCGCCTTACAAC-3'.

[0040] SEQ ID NO.9: 5'-TGTTATCCGCTCACAATTCCACAC-3'.

[0041] SEQ ID NO. 10: 5'-GGCAACACTTCGAGCGGTA-3'.

[0042] SEQ ID NO. 11: 5'-GACTTTTCTCAGCTTCAGCCAG-3'.

[0043] Table 1 PCR reaction procedure II. Imprinted Genes ZmMIE2 Genotyping of gene-edited strains 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.

[0044] 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.

[0045] 3. ZmMIE2 The two homologous chromosomes of C1ZmMIE2 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.

[0046] 4. ZmMIE2 The ZmMIE2 gene on both homologous chromosomes of C2 underwent the same mutation, specifically on both homologous chromosomes. Figure 1 The genes all have an insertion of one A base (730bp-731bp). ZmMIE2 B), which causes a frameshift, resulting in the loss of function of the protein ZmMIE2.

[0047] 5. Combination Figure 2 Gene expression profile data in maize ( ZmMIE2 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... Figure 3 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. ZmMIE2 Further research revealed that the core factor affecting grain area is a significant change in grain length.

[0048] Example 3 ZmMIE2 Results of gene protein structure and subcellular localization 1. For each ZmMIE2 In gene-edited mutants and overexpression lines, Figure 4 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. ZmMIE2 ).

[0049] 2. To further investigate the localization of this gene in cells, we conducted... ZmMIE2 Subcellular localization experiments of genes. Genes with correct sequencing... Figure 5 The purified CDS sequence product was ligated into the T-vector (GENSTAR, T184). ZmMIE2 The reaction system is as follows:

[0050] Table 2 Reaction systems for T-carrier linkage The prepared system was reacted at 37°C for 5 minutes.

[0051] 3, The connected product is directly converted into E. coli competent DH5a, and the conversion process is as follows: 1) The removed E. coli competent DH5a is thawed on ice; 2) Take an appropriate amount of ligation reaction solution and add it to the competent cells (the volume of the ligation reaction solution is ≤10% of the volume of the competent cells), mix gently, and ice bath for 30 min; 3) In a 42°C water bath for 2 min, then immediately placed in ice, ice bath for 2 min; 4) Add 500 μl of SOC medium, place in a 37°C shaker, 180 rpm, 1 h, activate E. coli; 5) The bacterial solution is evenly coated on an LB agar plate containing 50 μg / ml kanamycin, and cultured at 37°C for 12-16 h.

[0052] 4, Pick a single colony 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, place it in a 37°C shaker, 180 rpm, and culture for 3-4 h. Use M13 primers (use SEQ ID NO. 12 and SEQ ID NO. 13) to perform PCR on the bacterial solution, and send the bacterial solution to GenScript Biotech (Shanghai) Co., Ltd. for sequencing identification. Extract the plasmid with correct sequence and store it at -20°C for standby.

[0053] SEQ ID NO. 12: 5'-TGTAAAACGACGGCCAGT-3'; SEQ ID NO. 13: 5'-CAGGAAACAGCTATGACC-3'.

[0054] 5, Use KpnI and XbaI two endonucleases to digest the GFP empty vector: Table 3 KpnI and XbaI two endonuclease digestion system The reaction procedure is: 37°C, 1 h, 75°C, 15 min, and then use agarose gel DNA recovery kit (TIANGEN, DP210) for gel recovery to obtain the linearized GFP vector.

[0055] 6, Use primers containing homologous recombination specific sequences (ZmMIE2-GFP) to extract the ZmMIE2 plasmid as a template, and perform gel recovery after PCR amplification to obtain the CDS sequence of the ZmMIE2 gene with GFP homologous arms, which is used for subsequent ligation. Figure 6

[0056] ​7. Homologous recombination: homologous recombination was performed using the EZ-Flex Seamless Cloning Kit of GENSTAR (T197), and the reaction system was as follows: Table 4 Reaction system for homologous recombination Gently mix, react at 37°C for 30 min, and after the reaction is completed, place on ice. The product is directly transformed into E. coli competent DH5a, plated, picked, and cultured. Then, PCR identification is performed using a combination of specific primers of the gene (SEQ ID NO. 14) and specific primers of the GFP vector (SEQ ID NO. 15). The bacterial liquid with a band and correct position is sent to GenScript Biotech (Shanghai) Co., Ltd. for sequencing. The bacterial liquid with correct sequencing is subjected to plasmid extraction to obtain the recombinant GFP plasmid.

[0057] SEQ ID NO. 14: 5'-ACGGGGGACGAGCTCGATGGCAGCAGCCCCGGCGG-3'; SEQ ID NO. 15: 5'-GCTTCATGTGGTCGGGGTAGC-3'.

[0058] 8. Agrobacterium transformation 1) The competent GV3101 Agrobacterium (Weidi Biology, AC1002) taken out at -80°C is thawed on ice; 2) In a 1.5 ml centrifuge tube, 2 μl of the constructed recombinant GFP plasmid is added to each 50 μl of competent cells, and the mixture is placed on ice for 30 min; 3) The mixture is quickly frozen in liquid nitrogen for 5 min, and then is quickly transferred to ice for 5 min; 4) 500 μl of SOC medium is added to the centrifuge tube, and the mixture is incubated at 28°C, 200 rpm for 2-3 h; 5) 200 μl of the bacterial liquid is aspirated and spread on solid medium containing kanamycin antibiotic; 6) Invert the culture and incubate at 28°C in the dark for 48-72 h; 7) Prepare 2.0 ml sterile centrifuge tubes, add 1.5 ml of liquid medium containing kanamycin and rifampicin, and pick each single colony and place it in a new centrifuge tube. Incubate at 28°C, 200 rpm until the bacterial liquid is turbid (about 12 h); 8) Use specific primers of the gene (SEQ ID NO. 12) and specific primers of the GFP vector (SEQ ID NO. 13) to perform bacterial detection using the turbid bacterial liquid as a template to determine whether the plasmid is successfully transformed; 9) The transformed bacteria solution was taken 700 μl and mixed with 300 μl glycerol, and then placed in -80°C for long-term storage.

[0059] 9) Transient transformation of tobacco leaves 1) A 50 ml sterile centrifuge tube was prepared, 25 ml LB medium containing kanamycin and rifampicin was added, 100 μl Agrobacterium containing the plasmid to be transformed was added, and the mixture was incubated at 28°C, 200 rpm for 12 hours or more; 2) The concentration of the bacterial solution was determined by spectrophotometer, and the OD600 value was between 0.6 and 1.0; 3) Centrifugation at 3500 g for 15 min, discard the supernatant, repeat once to remove as much antibiotic as possible; 4) Finally, the bacterial solution was adjusted to OD600≈1 using resuspension solution; 5) At the same time, the Agrobacterium containing the P19 plasmid was also suspended and the concentration of the bacterial solution was adjusted to OD600≈1; 6) According to the amount of the final bacterial solution (3 mL is needed to completely wet each leaf), the bacterial solution containing the plasmid of interest (or marker plasmid) and the bacterial solution containing the P19 plasmid were mixed in equal volume in a new centrifuge tube, and the centrifuge tube was placed in 28°C, dark for 2-5 hours; 7) Select well-grown, healthy tobacco leaves (about 6-7 leaf age plants completely unfolded leaves) for Agrobacterium infection, water thoroughly 2-3 days before transformation, and control watering after transformation; 8) Before infection, place the tobacco under a white daylight lamp for 1 hour to open the stomata; 9) Before injection, resuspend the bacteria, and use a 1 ml syringe to take the bacterial solution; 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 the left hand, and gently press the syringe nozzle without the needle vertically on the back of the leaf with the right hand, slowly push with the right thumb, and observe the slow movement of the bacterial solution under the leaf epidermis until the whole leaf is infected, and label the injected petiole (2-3 leaves can be injected per plant, do not use cotyledons, if the leaf is large, 2-4 small holes can be made in different parts); 11) After injection, pay attention to keep the tobacco moist, and continue to culture for 2-5 days; 12) Prepare clean glass slides, drop a drop of ddH2O in the center of the glass slide, tear off the lower epidermis of the tobacco leaf and place it on the glass slide, and gently cover it with a cover glass from one side to avoid air bubbles; 13) Observe the fluorescence signal under a laser confocal microscope, find that the signal point is co-localized with the chloroplast marker, and determine that the ZmMIE2 protein is localized in the chloroplast (. ZmMIE2 ).

[0060] Example 4, ZmMIE2 Grain internal element determination of homozygous mutant lines and overexpression lines 1. Planting of materials (planting ZmMIE2 overexpression plants, ZmMIE2 frameshift mutant lines and wild type KN5585), ZmMIE2 overexpression and wild type self-crossing, ZmMIE2 frameshift mutant lines, ZmMIE2 frameshift mutant lines of different transformation events and wild type crossing and backcrossing, three plants for each inflorescence of each pollination method; take the inflorescences 15 days after pollination and at the mature stage to determine the 100-grain weight, protein, starch and soluble sugar content.

[0061] a. Determination of soluble sugar Stock solution preparation: 100 mg of glucose that has been dried to constant weight in an 80°C oven is prepared into a 1000 mL solution with 80% (by volume) ethanol, which is the glucose standard solution. 1 g of anthrone is dissolved in 1000 mL of dilute sulfuric acid solution to obtain anthrone reagent, which is placed in a brown bottle and prepared on the same day.

[0062] 1) Sample extraction Place the test grain in an oven at 110°C for 15 min, then adjust the temperature to 70°C and leave overnight. After the seeds are completely dried, grind them and weigh 0.05 g, then place it in a 10 ml centrifuge tube, add 4 ml of 80% ethanol, and place the sample in a 80°C water bath for 40 min of water bath treatment, stirring every 10 min. Then centrifuge at 5000g for 5 min, collect the supernatant. Add 0.01 g of activated carbon to the supernatant and decolorize at 80°C for 30 min. Finally, make up to 10 ml, and take the filtrate for determination.

[0063] 2) Draw the standard curve Prepare glucose solutions of different concentrations, add 5 ml of freshly prepared anthrone reagent, mix well, then place in a boiling water bath for 10 min, then quickly transfer to cold water and cool for 2 min. Measure the absorbance at a wavelength of 625 nm using a spectrophotometer. Draw the standard curve with glucose content as the abscissa and absorbance as the ordinate, and calculate the standard linear equation.

[0064] 3) Determination Take 1 ml of the filtrate from step 1) and mix with 5 ml of anthrone reagent. Determine the absorbance according to the same method in step 2), and determine the soluble sugar content according to the standard curve.

[0065] b. Determination of starch Take the residue produced in the first step of soluble sugar extraction with ethanol 0.5g, put into a flask, add 20g / l of HCl 25ml, boil in a boiling water bath for 3.5h, neutralize with 5mol / l NaOH, add Ba(OH)2 to precipitate completely, add a drop of phenolphthalein indicator. Add ZnSO4 while stirring. With the precipitation of barium salt, drop to red fade, and then add Ba(OH)2 solution to restore to light red as the end point. Filter, dilute to 250ml. Take 2ml to determine the glucose content.

[0066] Calculate the crude starch content = glucose content x 0.9 Protein determination c. Protein determination The Star G3100 near-infrared grain analyzer was used to ZmMIE2 Overexpression of self-fertilized grains, ZmMIE2 Frameshift mutation self-fertilized grains and wild type self-fertilized grains were determined, and each package of material should fill the determination column, knock the bottom of the determination column, so that the determination material fills the determination column without gap, determine three times, and process and analyze the data by Excel software.

[0067] d. Determination of 100-grain weight After air-drying ZmMIE2 Overexpression of self-fertilized ears, ZmMIE2 Frameshift mutation self-fertilized ears and wild type self-fertilized ears were threshed, and 100 grains were weighed for 100-grain weight, and 3 times were weighed for each ear, and 3 ears of each material were taken to calculate the 100-grain weight.

[0068] 2、 Figure 7 The results of the determination of the internal elements of the mutant and the wild type grain of the overexpression strain are shown in ZmMIE2 , ZmMIE2 The protein content of the mutant grain was significantly lower than that of the wild type, and the soluble sugar was significantly higher than that of the wild type. In addition to the related results of the overexpression strain, there were two groups of results of starch and 100-grain weight. Finally we can give a conclusion. That is ZmMIE2 Genes can affect the development of corn kernels, and overexpression ZmMIE2 of the gene can increase the kernel area and improve the 100-grain weight, which can be used to improve the yield of corn. And ​ when the gene is mutated, it can increase the protein content of the kernel, which lays a theoretical foundation for creating new varieties of high-protein corn.

[0069] Although the preferred embodiments of the present application have been described, those skilled in the art can make further changes and modifications to these embodiments once they know the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications falling within the scope of the present application.

Claims

1. Use of ZmMIE2 protein or its coding gene in regulating quality traits of corn kernels, characterized in that, The amino acid sequence of the ZmMIE2 protein is shown as SEQ ID NO.

2.

2. The use of ZmMIE2 protein or its encoding gene in regulating the quality traits of corn kernels according to claim 1, characterized in that, The nucleotide sequence of the coding gene is shown as SEQ ID NO.

1.

3. The use of ZmMIE2 protein or its encoding gene in regulating the quality traits of corn kernels according to claim 2, characterized in that, Promoting the expression of the ZmMIE2 protein to increase the grain area, hundred-grain weight and soluble sugar content of corn, and inhibiting the expression of the ZmMIE2 protein to increase the protein content of corn grain.

4. The use of ZmMIE2 protein or its encoding gene in regulating the quality traits of corn kernels according to claim 3, characterized in that, Promoting the expression of the ZmMIE2 protein is achieved by introducing a biological material containing the coding gene into corn.

5. The use of ZmMIE2 protein or its encoding gene in regulating the quality traits of corn kernels according to claim 4, characterized in that, The biological material is a recombinant vector, an expression cassette, a transgenic cell line or a recombinant bacteria.

6. The use of ZmMIE2 protein or its encoding gene in regulating the quality traits of corn kernels according to claim 3, characterized in that, Inhibiting the expression of the ZmMIE2 protein is achieved by gene editing through the CRISPR / Cas9 system.

7. The use of ZmMIE2 protein or its encoding gene in regulating the quality traits of corn kernels according to claim 6, characterized in that, The CRISPR / Cas9 system is a recombinant vector expressing gRNA and Cas9.

8. The use of ZmMIE2 protein or its encoding gene in regulating the quality traits of corn kernels according to claim 6, characterized in that, The target sequence of the gRNA is shown as SEQ ID NO.

5.

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

10. A method of breeding transgenic maize having increased grain protein content, comprising, It comprises the step of inhibiting the expression of the ZmMIE2 protein in corn as claimed in claim 1.

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