Application of ZmUUP5 protein in regulation and control of quality traits of corn kernels

Abstract

The invention belongs to the field of molecular biology, and discloses application of ZmUUP5 protein in regulation and control of corn kernel quality and yield traits. The amino acid sequence of the ZmUUP5 protein is as shown in SEQ ID NO. 2, and the nucleotide sequence of the ZmUUP5 protein coding gene is as shown in SEQ ID NO. 1. It is found that the ZmUUP5 protein can regulate and control the corn kernel quality and yield traits, mutation of the ZmUUP5 gene in corn can improve the protein content, overexpression of the ZmUUP5 gene can cause grain enlargement and hundred-grain weight increase, the ZmUUP5 protein can be used for improving the corn yield, and the content of amino acid and amylose in grains can also be improved. The discovery not only can provide a scientific basis for analyzing the development mechanism of grains, but also can provide theoretical guidance for genetic improvement of corn.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology, specifically the application of ZmUUP5 protein in regulating the quality traits of maize kernels. Background Technology

[0002] The phenomenon of genetic imprinting was first discovered simultaneously in mice and maize in the 1980s. Among them, genetic studies on endosperm traits in maize first revealed the uniqueness of plant genetic imprinting. Unlike animal imprinted genes, which mainly affect embryonic development, the functions of plant imprinted genes are highly related to the needs of human agricultural production. Their regulatory networks directly affect key agronomic traits such as crop yield formation, quality accumulation, and stress resistance, thus becoming a core research direction in the field of crop epigenetics breeding.

[0003] Imprinted genes are genes that, during the sexual reproduction of crops, undergo epigenetic modifications such as DNA methylation, histone modification, and non-coding RNA regulation, resulting in selective expression (only maternal or paternal expression) of homologous alleles from the father or mother. In major food crops such as maize, rice, and wheat, imprinted genes play a crucial role in regulating grain development. Their influence primarily focuses on three pathways: First, regulating grain structure formation by affecting endosperm cell differentiation, cell proliferation, and the coordinated development of the embryo and endosperm, thus determining grain size and morphology. Second, participating in nutrient accumulation by directly or indirectly regulating the synthesis rate and distribution ratio of major grain nutrients such as starch, protein, and oil, thus affecting crop quality. Third, regulating the developmental sequence by controlling the onset time, duration, and maturation process of grain filling, ensuring normal grain development and maturity, and avoiding yield losses due to abnormal development.

[0004] In maize, maternally expressed imprinted genes ZmMEG1 This gene is a typical example of regulating grain development. It is specifically expressed in the early stages of maize endosperm development. Its maternal allele promotes the formation and functional maintenance of transport cells by activating the differentiation signaling pathway of endosperm transport cells. Transport cells, as key channels for nutrient transport between the endosperm and maternal tissues, directly determine the efficiency of nutrient supply during the grain-filling period through their quantity and activity. Studies have shown that... ZmMEG1 The loss of gene function leads to a reduction in the number of endosperm cells in maize, a significant decrease in grain filling rate, and ultimately a reduction in 100-grain weight and starch content in the grains, fully demonstrating its core regulatory role in maize grain development and yield formation.

[0005] Besides imprinted genes, the F-BOX protein family genes are also important molecular nodes regulating crop grain development. As a key component of the ubiquitin-proteasome system, F-BOX proteins mediate the ubiquitination and degradation of target proteins by recognizing and binding to them, thereby regulating biological processes such as cell cycle, signal transduction, and metabolic pathways. The F-box domain was initially discovered in the cell cycle regulatory protein Cdc4, and its name originates from studies of cyclin F. The F-box domain consists of approximately 50 amino acid residues and has maintained a high degree of sequence conservation throughout evolution. Especially in the critical region where it binds to S-phasekinase-associated protein 1 (SKP1), a specific amino acid residue pattern exists. These conserved sequences are fundamental to maintaining its structural stability and functional specificity, ensuring that the F-box protein can form a stable interaction with SKP1, providing structural support for the subsequent assembly of the SCF (Skp1-Cullin-F-box protein) ubiquitin ligase complex. Based on the different types of domains in the C-terminal substrate-binding region of F-box proteins, they can be divided into several categories. Among them, FBW proteins containing the WD-40 domain form a β-propeller-like structure with its WD-40 repeat domain, providing a specific surface for substrate recognition; FBL proteins containing leucine-rich repeat sequences (LRR) form a horseshoe-shaped structure by the LRR domain, enabling highly specific substrate binding; while FBX proteins containing other different protein-protein interaction modules or no recognition motif have more diverse substrate binding modes and functional properties; in addition, there are some unique subfamilies of F-box proteins in plants, such as a class of F-box proteins unique to monocotyledons, which play a key role in plant-specific physiological processes.

[0006] In rice, OsFBK1 Genes have a significant impact on grain development. Compared to wild-type rice, osfbk1 The seeds of RNAi lines were significantly longer, while those of overexpressing plants were much longer. OsFBK1 The kernel width increases and the kernel weight rises. In corn, ZmFBL41 It is a typical F-BOX gene that regulates grain development: This gene is highly expressed during the grain-filling stage of maize grain development. It interacts with negative regulators in the abscisic acid (ABA) signaling pathway, mediates its ubiquitination and degradation, thereby enhancing the ABA signal response and promoting grain filling and dehydration maturation. ZmFBL41Loss of function leads to prolonged grain filling period and delayed dehydration in maize, which can easily cause grain mold, reduce the weight of 100 grains, and decrease the protein content in the grains. This indicates that both the F-BOX gene and the imprinted gene play irreplaceable roles in crop grain development, and there may be cross-synergy in their regulatory networks, providing multi-dimensional molecular targets for research on crop grain development regulation.

[0007] In summary, imprinted genes provide the core epigenetic regulatory basis for grain development by regulating grain structure formation, nutrient accumulation, and developmental timing. F-BOX genes, through ubiquitination-mediated signaling pathway regulation, become key molecular nodes for optimizing grain filling, ripening, and quality formation. Together, they constitute an important molecular network for regulating crop grain traits. However, among the currently identified imprinted and F-BOX genes, targets with the dual function of precisely regulating both grain nutritional quality and yield traits remain relatively scarce, making it difficult to meet the breeding needs for synergistic improvement in crop yield and quality. Summary of the Invention

[0008] Technical issue: The inventors previously discovered, through screening imprinted gene mutants in corn kernels, that the paternal imprinted gene... ZmUUP5 Gene mutations may affect kernel morphology and development. However, it is unknown whether the ZmUUP5 protein regulates kernel area, 100-kernel weight, amino acid content, starch content, and protein content in corn kernels.

[0009] Therefore, the first object of the present invention is to provide the application of the ZmUUP5 protein or its encoding gene in regulating maize kernel quality traits, wherein the amino acid sequence of the ZmUUP5 protein is shown in SEQ ID NO.2.

[0010] In preliminary research, the inventors successfully discovered a key gene with significant breeding value by conducting in-depth screening and precise identification of imprinted gene mutants in maize kernels. ZmUUP5 The gene encodes a product containing a conserved F-BOX domain, which can precisely participate in the regulation of target proteins through the ubiquitin-proteasome system. Furthermore, ZmUUP5 The gene's expression pattern closely matches the key stages of maize kernel development. The discovery of this gene opens up a completely new path in crop breeding, providing a highly promising and precise new target for overcoming the current technical bottlenecks in the synergistic breeding of crop imprinted genes and F-BOX genes. It is expected to bring about significant changes in high-yield and high-quality breeding for maize and even other crops.

[0011] Furthermore, the nucleotide sequence of the encoding gene is shown in SEQ ID NO.1.

[0012] Furthermore, promoting the expression of the ZmUUP5 protein can increase the kernel area, 100-kernel weight, amino acid content, and amylose content of corn kernels, while inhibiting the expression of the ZmUUP5 protein can increase the protein content of corn kernels.

[0013] Furthermore, the expression of the ZmUUP5 protein is promoted by transferring biological material containing the encoding gene into maize.

[0014] Furthermore, the biological material is a recombinant vector, expression cassette, transgenic cell line, or recombinant bacteria.

[0015] Furthermore, the inhibition of the expression of the ZmUUP5 protein was achieved through gene editing using the CRISPR / Cas9 system.

[0016] Furthermore, the CRISPR / Cas9 system is a recombinant vector expressing gRNA and Cas9.

[0017] Furthermore, the target sequence of the gRNA is shown in SEQ ID NO.5.

[0018] A second objective of this invention is to provide a method for cultivating transgenic maize with increased kernel area, 100-kernel weight, amino acid content, and amylose content, including a step of promoting the expression of the ZmUUP5 protein in maize.

[0019] 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 ZmUUP5 protein in maize.

[0020] The present invention has the following beneficial effects: The innovation of this invention lies in the fact that it reveals for the first time that corn ZmUUP5 Gene mutations can significantly increase the protein content of grains. Furthermore, ZmUUP5 Overexpression of this gene not only increases the surface area and 100-kernel weight of maize kernels, but also enhances the content of amino acids and amylose in the kernels. These findings provide a solid scientific foundation for a deeper understanding of the molecular mechanisms of maize kernel development and offer valuable theoretical guidance and practical direction for maize genetic improvement and breeding. Through these research results, we hope to cultivate new maize varieties with higher nutritional value and better yield performance, thereby making a significant contribution to agricultural production and food security. Attached Figure Description

[0021] Figure 1 for ZmUUP5 Identification of gene structure and frameshift mutant lines; A: ZmUUP5 B: Target sites for gene editing materials from gene overexpression lines; ZmUUP5Gene structure, mutation sites, and types.

[0022] Figure 2 for ZmUUP5 The expression profile of a gene obtained through quantitative fluorescence assay.

[0023] Figure 3 for ZmUUP5 Grain phenotypic analysis of gene frameshift mutant lines, overexpression lines, and wild-type lines; A: Wild-type and ZmUUP5 Phenotypic diagram of mature grain length and width of frameshift mutant lines; B: wild type and ZmUUP5 Phenotypic analysis of grain area in frameshift mutant lines; **: indicates highly significant at P<0.01.

[0024] Figure 4 for ZmUUP5 Grain phenotypic analysis of gene frameshift mutant lines, overexpression lines, and wild-type lines; A: Wild-type and ZmUUP5 Phenotypic analysis of grain width in frameshift mutant lines; B: Wild type and ZmUUP5 Phenotypic analysis of grain length in frameshift mutant lines; **: indicates highly significant at P<0.01.

[0025] Figure 5 Subcellular localization of the ZmUUP5 protein.

[0026] Figure 6 for ZmUUP5 Grain phenotypic analysis of frameshift mutant lines and wild-type lines in reciprocal crosses; A: ZmUUP5 Phenotypic diagram of grains from reciprocal crosses between frameshift mutant lines and wild-type lines; B: ZmUUP5 Grain area analysis of frameshift mutant lines and wild-type reciprocal crosses; **: indicates highly significant at P<0.01.

[0027] Figure 7 for ZmUUP5 Determination of seed indexes; A: 100-seed weight analysis of wild-type and frameshift mutant lines; B: Amino acid content analysis of wild-type and frameshift mutant lines; C: Starch content analysis of different types of wild-type and frameshift mutant lines; D: Protein content analysis of wild-type and frameshift mutant lines; **: indicates extremely significant at P<0.01. Detailed Implementation

[0028] 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.

[0029] Example 1: Imprinted Genes ZmUUP5 Cloning ZmUUP5 The nucleotide sequence of the gene is shown in SEQ ID NO.1. ZmUUP5 The amino acid sequence of the gene-encoded protein ZmUUP5 is shown in SEQ ID NO.2.

[0030] SEQ ID NO.1:

[0031] SEQ ID NO.2: MAETTLLTRRSKRIALRVSLAGAKPSWRDWAGLAPLAVEKIADRLRDEDVADFMRFRLVCKLWMDGSGIRKPRELGARVIDHSFHPRRWILLTDEKKEADEATPTRRMLLNLTTRKTIQVDLPELAGHSVVPGPAAAPEGMLVVRDERSLVVRLLNPLTRHVVDLPTLLTLRPGNRRRGPVPSSFAEDHEVTAAGFADASTI VVYLGNANQLVVARPGDARWTLVGGLDPEFPLRSTATFQSRFYCVDRCQLLVVDMERGPRAQLVVAANLERRYRTVGMVDDGGRLMAVCSRDQVVRADARWMSSLETRVELFHVDLQEEKLSRIEDLGERAVFAGLRGAVLLPSTKYYFSVDRGTVFFRFGSSQRHFGAFHVRRRHTCYIASVWGKLPQRVASYVTTLRYLY.

[0032] Example 2, Gene ZmUUP5 Overexpression and construction of gene-edited lines 1. Genes ZmUUP5 Construction of overexpression lines: 1) Maize genome annotation database MaizeGDB was retrieved. ZmUUP5 The complete nucleotide sequence of the gene, as shown in SEQ ID NO.1, was extracted. ZmUUP5 RNA from specifically expressed wild-type KN5585 tissue was extracted and reverse transcribed to obtain cDNA. The cDNA was then amplified by PCR using the sequences SEQ ID NO.3 and SEQ ID NO.4 and purified to obtain... ZmUUP5 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'-ATGGCTGAGACGACCCTGCTGACGA-3'.

[0034] SEQ ID NO. 4: 5'-CTAGTAGAGATAACGGAGGGTGGTGA-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. ZmUUP5 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 ZmUUP5 Construction of gene editing vectors: (1) First choose to be close to ZmUUP5 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).

[0039] SEQ ID NO.5: 5′CTCTGCGCGTTTCCCTTGC3′ (i.e., bits 44-62 of SEQ ID NO.1 from the end of 5′).

[0040] SEQ ID NO. 6: 5'-ACTCCAAGCCCTACAAACCAC-3'.

[0041] SEQ ID NO. 7: 5'-GTCCATCCACAGCTTGCACACC-3'.

[0042] (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.

[0043] (3) Using 10x 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℃.

[0044] Example 3, Imprinted Genes ZmUUP5 Genotyping of gene-edited mutant lines and overexpression lines I. Imprinted genes ZmUUP5 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, the procedure is shown in Table 1) to screen for overexpression-positive plants.

[0045] SEQ ID NO. 8: 5'-GCAAAGTCTGCCGCCTTACAAC-3'.

[0046] SEQ ID NO.9: 5'-TGTTATCCGCTCACAATTCCACAC-3'.

[0047] Table 1 PCR reaction procedure II. Imprinted Genes ZmUUP5 Genotyping of gene-edited strains 1. Using genomic DNA from maize leaves of gene-edited strains as templates, the gene sequence approximately 500 bp near the target site was amplified by PCR using primers consisting of SEQ ID NO.6 and SEQ ID NO.7 (the procedure is shown in Table 1), and the corresponding PCR amplification products were obtained.

[0048] 2. Perform Sanger sequencing on the PCR amplification products. Sequencing results and... ZmUUP5 The target sequences of genes edited by the CAS9 protein were compared to select homozygous mutation types in the target site regions.

[0049] 3. ZmUUP5- The two homologous chromosomes of C1 ZmUUP5 The same mutation occurred in the genes, specifically on two homologous chromosomes. ZmUUP5 All genes have a deletion of 8 bases (54bp-61bp). Figure 1 B), which causes a frameshift, resulting in the loss of function of the protein ZmUUP5.

[0050] 4. ZmUUP5-The ZmUUP5 gene on both homologous chromosomes of C2 underwent the same mutation, specifically on both homologous chromosomes. ZmUUP5 All genes have a deletion of 26 base pairs (34bp-60bp). Figure 1 B), which causes a frameshift, resulting in the loss of function of the protein ZmUUP5.

[0051] 5. Combination ZmUUP5 Gene expression profile data in maize ( Figure 2 The gene was found to be highly expressed in grains, so it was targeted... ZmUUP5 The grain area, grain width, and grain length of the gene mutants and overexpression lines were measured. The results are as follows: Figure 3 and Figure 4 As shown, two ZmUUP5 The seed area, seed width, and seed length of the gene-edited lines were significantly smaller than those of the wild type, while the seed area of ​​the overexpression lines was significantly larger than that of the wild type.

[0052] Example 4: Subcellular localization results of ZmUUP5 protein 1. To investigate the cellular localization of this gene, we conducted a subcellular localization experiment of the ZmUUP5 protein. (The sequenced protein will be...) ZmUUP5 The purified CDS sequence product of the gene was ligated into the T-vector (GENSTAR, T184). The reaction system is as follows:

[0053] Table 2 Reaction systems for T-carrier linkage The prepared system was reacted at 37°C for 5 minutes.

[0054] 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; 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; 3) In a 42°C water bath for 2 minutes, then immediately place in ice for 2 minutes; 4) Add 500 μl of SOC medium and place in a shaker at 37°C, 180 rpm, for 1 h to activate E. coli; 5) Spread the bacterial suspension evenly onto LB agar plates containing 50 μg / ml kanamycin and incubate at 37°C for 12-16 h.

[0055] 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 (SEQ ID NO.10 and SEQ ID NO.11). 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.

[0056] SEQ ID NO. 10: 5'-TGTAAAACGACGGCCAGT-3'.

[0057] SEQ ID NO. 11: 5'-CAGGAAACAGCTATGACC-3'.

[0058] 5. Enzyme digestion: The empty GFP vector was digested using two restriction enzymes, KpnI and XbaI. Table 3. Enzyme digestion systems of KpnI and XbaI endonucleases 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.

[0059] 6. Using primers (ZmUUP5-GFP) containing homologous recombination-specific sequences, the extracted... ZmUUP5 Using the plasmid as a template, PCR amplification was followed by gel recovery to obtain plasmids with added GFP homologous arms. ZmUUP5 The CDS sequence is used for subsequent connections.

[0060] 7. Homologous recombination: Homologous recombination was performed using GENSTAR's EZ-Flex Seamless Cloning Kit (T197). The reaction system is as follows: Table 4 Reaction systems for homologous recombination 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.12) and GFP vector-specific primers (SEQ ID NO.13). Bacterial solutions with bands in the correct positions were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. Plasmids were extracted from the sequencing solutions.

[0061] SEQ ID NO. 12: 5'-ATCCTGCTGACGGATGAGAAGAAGGAGG-3'.

[0062] SEQ ID NO. 13: 5'-GCTTCATGTGGTCGGGGTAGC-3'.

[0063] 8. Agrobacterium-mediated transformation 1) Thaw competent cells of GV3101 Agrobacterium (Video Biotechnology, AC1002) taken out at -80℃ on ice; 2) Add 2 μl of the constructed vector plasmid to every 50 μl of competent cells in a 1.5 ml centrifuge tube and incubate on ice for 30 min; 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; 4) Add 500 μl of SOC medium to the centrifuge tube, incubate at 28℃ and 200 rpm for 2-3 hours to recover; 5) Take 200 μl of bacterial suspension and spread it on a solid culture medium containing kanamycin antibiotic; 6) Invert the container and incubate at 28°C in the dark for 48-72 hours; 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). 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; 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.

[0064] 9. Instantaneous conversion of tobacco leaves 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. 2) The concentration of the bacterial culture was determined using a spectrophotometer, with an OD600 value between 0.6 and 1.0; 3) Centrifuge at 3500g for 15 minutes, discard the supernatant, and repeat once to remove antibiotics as much as possible; 4) Finally, adjust the bacterial suspension to OD600≈1 using the resuspension solution; 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; 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. 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. 8) Before inoculation, place the tobacco under a white fluorescent light for 1 hour to allow the pores to open; 9) Before injection, resuspend the bacterial cells and draw up the bacterial solution with a 1ml syringe; 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). 11) Keep the tobacco moist after injection and continue culturing for 2-5 days; 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. 13) Observing the fluorescence signal under a laser confocal microscope, the green fluorescence signal of p1300-ZmUUP5-GFP co-localizes with the signal site in the Golgi apparatus within the cell, indicating that this protein is localized in the Golgi apparatus. Figure 5 ).

[0065] Example 5 ZmUUP5 Elemental analysis of grains from homozygous mutant lines and overexpression lines 1. Material planting (planting) ZmUUP5 overexpression plants, ZmUUP5 Frameshift mutant lines and wild-type KN5585). ZmUUP5 Overexpression and wild-type self-pollination, ZmUUP5 Frameshift mutant lines ZmUUP5 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 kernels, protein and starch content were measured for the ears 15 days after pollination and at maturity.

[0066] a. Determination of starch Place the seeds to be tested 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 0.5g into an Erlenmeyer flask. Add 25ml of 20g / L HCl and boil in a water bath for 3.5h. Neutralize with 5mol / 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 Ba(OH)2 solution to restore the pale red color as the stop point. Filter and dilute to 250ml. Take 2ml to determine the glucose content.

[0067] Calculate crude starch content = glucose content × 0.9; protein determination. b. Protein determination Using a Starlink G3100 near-infrared grain analyzer to analyze grains ZmUUP5 Overexpression of self-pollinated seeds ZmUUP5 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.

[0068] c. Determination of 100-grain weight After air drying ZmUUP5 Self-pollinated fruit ears of overexpression lines ZmUUP5 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.

[0069] 2. ZmUUP5 Grain phenotypic analysis of frameshift mutant lines and wild-type reciprocal crosses is as follows: Figure 6 As shown, the results indicate that the wild type was used as the female parent. Zmuup5 The seed area of ​​the mutant was significantly smaller than that of the reciprocal cross when used as the male parent, and this was confirmed in both mutant combinations. This phenotype is also consistent with... ZmUUP5 Phenotypic patterns of genes as paternal imprinted genes.

[0070] Figure 7 Showing ZmUUP5 Comparison of 100-kernel weight and internal elemental content among gene mutants, overexpression lines, and wild-type maize kernels. Analysis showed that... ZmUUP5 The mutant maize had significantly lower kernel weight, amino acid content, and amylose content than wild-type maize, but significantly higher protein content. Conversely, ZmUUP5 The overexpressing strain had significantly higher grain weight, amino acid content, and amylose content than the wild type, but significantly lower protein content.

[0071] Based on these findings, we can conclude that: ZmUUP5 Genes play a crucial role in regulating maize kernel development. The function of this gene not only affects kernel weight and composition but may also have a potential positive impact on increasing maize yield and protein content. Therefore, ZmUUP5 Gene research provides important theoretical basis and practical guidance for breeding new maize varieties with high yield and high protein content.

[0072] 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.

[0073] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. The application of ZmUUP5 protein or its encoding gene in regulating maize kernel quality traits, characterized in that, The amino acid sequence of the ZmUUP5 protein is shown in SEQ ID NO.

2.

2. The application of the ZmUUP5 protein or its encoding gene according to claim 1 in regulating maize kernel quality traits, characterized in that, The nucleotide sequence of the encoding gene is shown in SEQ ID NO.

1.

3. The application of the ZmUUP5 protein or its encoding gene according to claim 2 in regulating maize kernel quality traits, characterized in that, Promoting the expression of the ZmUUP5 protein increases the kernel area, 100-kernel weight, amino acid content, and amylose content of corn kernels, while inhibiting the expression of the ZmUUP5 protein increases the protein content of corn kernels.

4. The application of the ZmUUP5 protein or its encoding gene according to claim 3 in regulating maize kernel quality traits, characterized in that, The expression of the ZmUUP5 protein is promoted by transferring biological material containing the encoding gene into maize.

5. The application of the ZmUUP5 protein or its encoding gene according to claim 4 in regulating maize kernel quality traits, characterized in that, The biological material is a recombinant vector, expression cassette, transgenic cell line, or recombinant bacteria.

6. The application of the ZmUUP5 protein or its encoding gene according to claim 3 in regulating maize kernel quality traits, characterized in that, Inhibition of the expression of the ZmUUP5 protein was achieved through gene editing using the CRISPR / Cas9 system.

7. The application of the ZmUUP5 protein or its encoding gene according to claim 6 in regulating maize kernel quality traits, characterized in that, The CRISPR / Cas9 system is a recombinant vector expressing gRNA and Cas9.

8. The application of the ZmUUP5 protein or its encoding gene according to claim 6 in regulating maize kernel quality traits, characterized in that, The target sequence of the gRNA is shown in SEQ ID NO.

5.

9. A method for cultivating transgenic maize with improved kernel area, 100-kernel weight, amino acid content, and amylose content, characterized in that, Includes the step of promoting the expression of the ZmUUP5 protein as described in claim 1 in maize.

10. A method for cultivating transgenic maize with increased grain protein content, characterized in that, Includes the step of inhibiting the expression of the ZmUUP5 protein as described in claim 1 in maize.

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