Use of biologicals of ZmTHX12 in improving corn kernel quality and yield

Abstract

This invention belongs to the field of molecular biology technology, specifically relating to a... ZmTHX12 The application of biological products in improving corn kernel quality and yield, the aforementioned ZmTHX12 The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2. This invention discovers... ZmTHX12 It can regulate the quality and yield traits of corn kernels, and in corn... ZmTHX12 Gene mutations can increase the protein and soluble sugar content in kernels, and can be used to improve the quality traits of corn; while ZmTHX12 ZmTHX12 Gene overexpression leads to increased oil and amylopectin content in kernels, resulting in larger kernels and increased 100-kernel weight, which can be used to improve maize yield and quality traits. 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.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology technology, specifically relating to a... ZmTHX12 The application of biological products in improving the quality and yield of maize kernels. Background Technology

[0002] The core characteristic of imprinted genes is that the expression pattern of parental alleles in offspring breaks the equal expression pattern prescribed by Mendel's laws of inheritance. Only the maternal or paternal allele is effectively expressed, while the other is silent or expressed at very low levels. In the sexual reproduction process of angiosperms, their genome consists of two maternal genomes and one paternal genome. Based on expression preferences, they can be divided into maternally expressed genes and paternally expressed genes. Maternally expressed genes, that is, in hybrid offspring, the expression level of maternal alleles is significantly higher than that of paternal alleles; paternally expressed genes, on the other hand, are expressed in the opposite way.

[0003] The regulation of imprinted gene expression in plants relies on the synergistic interaction of multiple epigenetic modifications. Taking the model plant Arabidopsis thaliana as an example, the activation of maternally imprinted genes hinges on the action of the DNA demethylase DME. This enzyme is specifically expressed in the centrocellular region of the female gametophyte, relieving transcriptional repression by targeting and removing methylation markers from the promoter regions of maternal alleles, thereby ensuring the preferential expression of maternally imprinted genes in the endosperm. Simultaneously, imprinted genes play a central role in plant growth and development, particularly seed development. They not only provide necessary nutrient storage space and structural support for embryo development by regulating the cellularization process and differentiation pattern of the endosperm, but also directly participate in the metabolic allocation of energy storage substances within the seed. For example, maternally expressed genes in Arabidopsis thaliana... Fertilisation-independent seed 2 It can influence endosperm development by regulating auxin signaling, thereby altering seed size; while paternally expressed genes in maize... maternally expressed gene 1 These genes participate in nutrient transfer in the endosperm, and their expression levels are directly related to grain filling efficiency and final grain weight. Furthermore, some imprinted genes remain expressed during seed germination, ensuring the orderly initiation of the germination process by regulating the balance of gibberellin and abscisic acid. In breeding practice, most imprinted genes, including those mentioned above, can only achieve "single-point improvement" of the grain, making it difficult to simultaneously optimize multiple quality indicators to meet production needs. Therefore, discovering and utilizing new imprinted genes or dominant expression genes has become an important direction for improving maize grain traits. Summary of the Invention

[0004] In the early stages of this invention, through screening imprinted gene mutants in maize kernels, it was discovered that the paternal imprinted gene... ZmTHX12 Gene mutations can affect the morphological development of grains. However, ZmTHX12Whether genes regulate kernel area, corn kernel oil content, corn kernel amylopectin content, corn kernel soluble sugar content, and protein content is unknown.

[0005] The technical solution adopted in this invention is: This invention provides ZmTHX12 The application of biological products in improving corn kernel quality and yield, the aforementioned ZmTHX12 The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2.

[0006] Preferably, the biological product is a formulation containing any one or more substances from A to C below: A: Homologous nucleic acid molecules with the nucleotide sequence shown in SEQ ID NO.1; specifically, recombinant vectors or recombinant bacteria containing A; B: A homologous amino acid sequence to the amino acid sequence shown in SEQ ID NO.2; specifically, a recombinant vector or recombinant bacterium containing B; C: Substances that can alter the expression levels of A and / or B.

[0007] Preferably, the homologous nucleic acid molecule refers to a nucleic acid molecule that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology with SEQ ID NO.1. The homologous amino acid sequence refers to an amino acid sequence that has at least 60%, at least 65%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology with SEQ ID NO.2.

[0008] Preferably, the substance capable of altering the expression levels of A and / or B includes any one of the following I and II: I: Substances that can increase the expression levels of substances A and / or B; II: Substances that can reduce the expression levels of substances A and / or B.

[0009] Preferably, I is selected from recombinant vectors containing homologous nucleic acid molecules, expression cassettes, transgenic cell lines, transgenic plant tissues, or recombinant bacteria; II is a CRISPR / Cas9 system for knocking out homologous nucleic acid molecules, the CRISPR / Cas9 system comprising a vector for expressing gRNA and a vector for Cas9.

[0010] Preferably, the sequence of the gRNA is shown in SEQ ID NO.5.

[0011] Preferably, the starting vector for expressing the gRNA vector is pBUE411.

[0012] Preferably, the starting vector of the recombinant vector containing homologous nucleic acid molecules is pCambia1300.

[0013] Preferably, I is used to improve the kernel shape, oil content, amylopectin content and yield of corn kernels; II is used to improve the protein content and soluble sugar content of corn kernels.

[0014] Preferably, the kernel shape of corn includes kernel length, kernel width, and kernel area.

[0015] Compared with the prior art, the beneficial effects of the present invention are: This invention provides ZmTHX12 The application of biological products in improving corn kernel quality and yield, the aforementioned ZmTHX12 The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2. This invention is the first to discover [a specific gene] in maize. ZmTHX12 Gene mutations can increase the protein and soluble sugar content of corn kernels; ZmTHX12 Gene overexpression can increase corn oil content, amylopectin content, kernel area, and 100-kernel weight. Unlike most imprinted genes that regulate only a single trait, ZmTHX12 Genes and related biological products can simultaneously affect multiple key quality indicators such as protein, soluble sugar, oil content, amylopectin, grain weight, and grain shape, breaking through the limitations of traditional "single-point improvement" and providing a new technical approach for breeding maize varieties with excellent comprehensive traits.

[0016] This invention also discloses ZmTHX12 Genes play a dual role in regulating grain morphology and nutrient accumulation. Mutations and overexpressions of genes lead to different directions of quality optimization, providing important clues for further analysis of the genetic and epigenetic regulatory network of grain development. This invention not only provides a scientific basis for understanding the mechanisms of grain development but also offers theoretical guidance for the genetic improvement of maize. Attached Figure Description

[0017] Figure 1 for ZmTHX12 Identification of gene structure and frameshift mutant lines; A: ZmTHX12 Construction of overexpression recombinant vectors; B: ZmTHX12 Target sites for gene editing; C: ZmTHX12 Gene structure, mutation sites, and types.

[0018] Figure 2 To obtain by quantitative fluorescence experiment ZmTHX12 Gene expression profile. In the diagram, EM represents the embryo; En represents the endosperm; and S represents the seed.

[0019] Figure 3 for ZmTHX12 Phenotypic analysis of the seeds of gene-edited lines, overexpression lines, and wild-type lines; A: Phenotypic diagram of mature seed length and width of different plants; B: Analysis of 100-seed weight of different plants; C: Phenotypic analysis of seed area of ​​different plants; D: Phenotypic analysis of seed length of different plants; E: Phenotypic analysis of seed width of different plants; **: indicates extremely significant at P<0.01.

[0020] Figure 4 for ZmTHX12 Embryo phenotype analysis of gene-edited lines, overexpression lines, and wild-type lines; A: Phenotypic diagram of mature embryo length and width of different plants; B: Phenotypic analysis of embryo area of ​​different plants; C: Phenotypic analysis of embryo length of different plants; D: Phenotypic analysis of embryo width of different plants; **: indicates extremely significant at P<0.01.

[0021] Figure 5 for ZmTHX12 Determination of grain parameters in gene-edited lines, overexpression lines, and wild-type plants; A: Protein content analysis of different plants; B: Oil content analysis of different plants; C: Amylopectin content analysis of different plants; D: Soluble sugar content analysis of different plants; **: indicates extremely significant at P<0.01.

[0022] Figure 6 for ZmTHX12 Genetic phylogenetic tree. A: ZmTHX12 Gene phylogenetic tree; B: ZmTHX12 Gene domain analysis.

[0023] Figure 7 for ZmTHX12 Gene α-sheet prediction.

[0024] Figure 8 Predictive analysis of the Pfam domain of ZmTHX12 protein Detailed Implementation

[0025] The present invention will be further illustrated below with specific embodiments, but these embodiments do not limit the scope of the invention. Modifications or substitutions to the details and form of the technical solutions of the present invention may be made without departing from the spirit and scope of the invention, but all such modifications or substitutions fall within the protection scope of the present invention.

[0026] The inventive concept of this invention is as follows: The study of plant imprinted genes is of irreplaceable importance for a deeper understanding of plant growth and development patterns and for revealing the genetic essence of species. However, this research field still faces many key scientific and technological challenges. First, there is a lack of systematic and comprehensive understanding of the expression regulatory networks and dynamic changes of imprinted genes in different species, tissues, organs, and developmental stages, and their impact on regulatory processes. Second, there is still a lack of mature and reliable technical routes and application strategies for effectively applying existing research results on plant imprinted genes to crop genetic improvement practices, and for precisely regulating their expression to optimize agronomic traits. Therefore, in-depth analysis of the regulatory mechanisms of plant imprinted genes and active exploration of their diverse applications in agricultural production constitute the core starting point and key objective of this invention.

[0027] Based on this, the present invention provides an application of imprinted genes in maize genetic improvement, namely... ZmTHX12 Application in improving maize kernel quality traits. This invention utilizes phylogenetic analysis of the amino acid sequence of ZmTHX12, revealing that the ZmTHX12 protein is conserved in monocotyledonous plants, and that a homologous protein with 90% homology exists in maize. Domain prediction indicates that this protein contains both a GT1 domain and a PRK14951 domain.

[0028] The GT1 domain typically contains a conserved sequence that binds to specific cis-acting elements. Located in a specific functional region of the relevant transcription factor, it exhibits high structural conservatism across plant species. This domain is topologically similar to the DNA-binding domain of the Myb protein, but with a longer α-helix. The GT1 domain is a domain that combines DNA binding and protein regulation functions; it can directly bind to Box II cis-elements in the promoter region of light-responsive genes and can also bind via Ca2+. 2+ Phosphorylation / dephosphorylation modifications regulate their own activity, thereby participating in plant transcriptional regulation. For example, the GT-1 protein in Arabidopsis thaliana can regulate the expression of downstream light-responsive genes through this domain in response to light signals. In rice, the GT1 domain regulates grain filling and final morphology by participating in the auxin signaling pathway. Researchers constructed... osgt1::atyucca1:gus The expression vector showed specific expression in the endosperm of rice grains, and the auxin response reporter gene in this transgenic line was also observed. dr5:gfp The expression level of the GT1 domain was significantly higher than that of the wild type, indicating that proteins containing the GT1 domain can affect the hormonal microenvironment during endosperm development by regulating the expression of auxin synthesis-related genes. As the core site for the accumulation of storage substances in grains, the developmental state of the endosperm directly determines grain weight and quality. This suggests that the GT1 domain may participate in the regulation of substance accumulation during the grain-filling period by mediating auxin signaling.

[0029] PRK14951 is primarily found in the γ and τ subunits of bacterial DNA polymerase III. Both are produced by the same gene through post-translational processing or selective expression, exhibiting highly similar sequences and containing this domain. The τ subunit is longer and contains an additional region that interacts with the core subunit. This domain is approximately 200-300 amino acids long and contains conserved Walker A and B motifs, which are involved in ATP binding and hydrolysis, respectively. These motifs are highly conserved in Gram-negative and Gram-positive bacteria. Its core function is related to ATPase activity and protein-protein interactions: the γ subunit acts as a clamp loader core, driving the loading and unloading of the clamp onto the DNA through its ATPase activity, enhancing the stability of polymerase binding to the template; the τ subunit, in addition to retaining similar functions, has a C-terminal extension that can bind to the α subunit of the polymerase core subunit and the helicase DnaB, acting as a replicon linker to coordinate the co-synthesis of the leading and lagging strands. The PRK14951 domain, by mediating these processes, is a key guarantee for efficient bacterial DNA replication.

[0030] To enable those skilled in the art to better understand and implement the technical solutions of this invention, the invention will be further described below with reference to specific embodiments. Unless otherwise specified, all reagents used in this invention are commercially available, and all methods used are conventional techniques in the art.

[0031] Example 1 ZmTHX12 The application of biological products in improving the quality and yield of maize kernels is as follows: 1. Imprinted genes ZmTHX12 Clones.

[0032] ZmTHX12 The nucleotide sequence of the gene is shown in SEQ ID NO.1. ZmTHX12 The gene encodes the protein ZmTHX12, the amino acid sequence of which is shown in SEQ ID NO.2.

[0033] SEQ ID NO.1:

[0034] SEQ ID NO.2: .

[0035] 2. ZmTHX12 Construction of gene overexpression lines and gene-edited lines.

[0036] (1) Gene ZmTHX12 Construction of overexpression lines.

[0037] 1) Maize genome annotation database MaizeGDB was retrieved.ZmTHX12 The complete nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0038] Extraction of wild-type KN5585 maize plants ZmTHX12 RNA was specifically expressed from the tissue, and cDNA was obtained by reverse transcription. The cDNA was then amplified by PCR using the sequences SEQ ID NO.3 and SEQ ID NO.4, and purified to obtain... ZmTHX12 The CDS sequence was obtained, and the PCR purified product was sent to Shanghai Sangon Biotech Co., Ltd. for sequencing.

[0039] SEQ ID NO.3: 5'-ATGCAGCAGCACCACCAGGGTGGCG-3'; SEQ ID NO. 4: 5'-CTATTGAACCATGGCGAGGAAGGT-3'.

[0040] 2) Amplification was performed using a high-fidelity enzyme, and the PCR product with the correct bands was recovered by gel electrophoresis. The pCambia1300 vector was linearized using restriction enzymes KpnI and XbaI. ZmTHX12 The CDS region of the gene is recombinated with a linearized pCambia1300 vector to obtain an overexpression recombinant vector, such as... Figure 1 As shown in A, it was then transferred into Escherichia coli DH5α strain.

[0041] 3) After the selected single colonies are tested, they are sent to the company for sequencing.

[0042] 4) Take the strain with correct sequencing results, extract the plasmid according to the steps shown in the TIANGEN plasmid rapid extraction kit, and store it in a -20℃ refrigerator for later use.

[0043] 5) The overexpression recombinant vector is introduced into maize and screened to obtain the overexpression line.

[0044] (2) ZmTHX12 Construction of gene-edited lines.

[0045] 1) such as Figure 1 As shown in B, first select the area closest to ZmTHX12 A 19bp target sequence was designed for the CDS region of the gene translation start codon, as shown in SEQ ID NO.5; primers for the target sequence were synthesized, as shown in SEQ ID NO.6 and SEQ ID NO.7. 2) Amplification was performed using the intermediate vector pCBC-MT1T2 as a template, and the PCR product containing the target sequence, i.e., gRNA, was purified and recovered.

[0046] pCBC-MT1T2 is disclosed in Xing HL, Dong L, Wang ZP, Zhang HY, Han CY, Liu B, Wang XC, Chen QJ. A CRISPR / Cas9 toolkit for multiplex genome editing inplants. BMC Plant Biol. 2014 Nov 29; 14:327.

[0047] 3) Using 10×BsaI restriction enzyme and high-concentration T4 ligase, the PCR product and pBUE411 vector were digested and ligated, transformed into E. coli and single clones were selected for identification. The correctly ligated single clones were selected, cultured and the plasmid was extracted and stored at -20℃ to obtain the gene editing vector.

[0048] SEQ ID NO.5: 5′ACGCTCAAAGGCCCCCTAT3′, which is bits 861 to 879 of SEQ ID NO.1 starting from the end of 5′.

[0049] SEQ ID NO.6: 5'-CAATTCAGTACCAGTACTAGCAAA-3'; SEQ ID NO. 7: 5'-ATTGGGCGCAGCGAGCGACGCAG-3'.

[0050] 4) Using the CRISPR / Cas9 system to analyze corn ZmTHX12 Genes were knocked out to obtain gene-edited strains.

[0051] 3. Imprinted genes ZmTHX12 Grain phenotype identification of overexpression lines and gene-edited lines.

[0052] (1) Imprinted genes ZmTHX12 Genotyping of overexpression lines.

[0053] Using genomic DNA from leaves of T0 generation overexpression lines as templates, the specific Bar gene sequence on the overexpression recombinant vector was amplified using SEQ ID NO.8 and SEQ ID NO.9, as shown in Table 1, to screen for positive overexpression plants.

[0054] SEQ ID NO.8: 5'-GCAAAGTCTGCCGCCTTACAAC-3'; SEQ ID NO.9: 5'-TGTTATCCGCTCACAATTCCACAC-3'.

[0055] Table 1 PCR reaction procedure Note: In Table 1, " / " indicates that this item is not present.

[0056] (2) Imprinted genes ZmTHX12 Genotyping of gene-edited strains.

[0057] 1) Using genomic DNA from maize leaves of gene-edited strains as templates, the gene sequence within 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.

[0058] 2) Perform Sanger sequencing on the PCR amplification products. Sequencing results and... ZmTHX12 The target sequence of the gene edited by the CAS9 protein was compared, and mutants with homozygous target site regions were selected. Plants with two mutant types were obtained and denoted as [missing information]. ZmTHX12- C1 and ZmTHX12- C2.

[0059] ZmTHX12- The two homologous chromosomes of C1 ZmTHX12 The same mutation occurred in the genes, specifically on two homologous chromosomes. ZmTHX12 There is a one-base deletion at 367bp of the gene, see Figure 1 The C-value causes a frameshift, resulting in the loss of function of the ZmTHX12 protein. ZmTHX12- The two homologous chromosomes of C2 ZmTHX12 The same mutation occurred in the genes, specifically on two homologous chromosomes. ZmTHX12 There is a 5-base deletion between 363bp and 367bp of the gene, see... Figure 1 The C-value causes a frameshift, resulting in the loss of function of the ZmTHX12 protein.

[0060] Combination ZmTHX12 Gene expression profile data in maize, see Figure 2 They discovered that this gene was highly expressed in grains, so they subsequently targeted... ZmTHX12 The grain area, grain width, and grain length of the gene-edited and overexpressed lines were measured.

[0061] The results showed that, Figure 3 As shown, two ZmTHX12 Gene-edited strains THX12- m1 and THX12 The grain area of ​​the -m2 strain was significantly smaller than that of the wild type, while the overexpression lines THX12 -OE1 and THX12-OE2 has a significantly larger grain area than the wild type, as shown in the results. Figure 3 .against ZmTHX12 Embryo area, embryo width, and embryo length, among other traits, were measured in gene-edited and overexpression lines. The results showed that... Figure 4 As shown, two ZmTHX12 The embryo area of ​​gene-edited lines was significantly smaller than that of wild-type lines, while the embryo area of ​​overexpression lines was significantly larger than that of wild-type lines. (See...) Figure 4 .

[0062] 4. ZmTHX12 Elemental analysis of the grains of self-crossed progeny of gene-edited and overexpression lines.

[0063] (1) Planting ZmTHX12 overexpression plants, ZmTHX12 The protein content, 100-grain weight, oil content, soluble sugar content, and amylopectin content of mature ears of the frameshift mutant line and the wild-type KN5585 were determined.

[0064] (2) Measurement method.

[0065] Protein determination: A Starlink G3100 near-infrared grain analyzer was used for analysis. ZmTHX12 Overexpression of self-pollinated seeds ZmTHX12 The self-pollinated seeds of the gene-edited strain and the self-pollinated seeds of the wild type were tested. During the test, each package of material should be filled to the top of 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.

[0066] Determination of the weight of 100 grains: After air drying ZmTHX12 Self-pollinated fruit ears of overexpression lines ZmTHX12 After threshing the self-pollinated ears of the gene-edited strain and the wild-type self-pollinated ears, 100 kernels were taken from each strain and weighed to determine the weight of 100 kernels. Each ear was weighed three times, and three ears of each material were taken to calculate the weight of 100 kernels.

[0067] Determination of oil content: After air drying ZmTHX12 Self-pollinated fruit ears of overexpression lines ZmTHX12 After threshing, 100 kernels were collected from each of the self-pollinated ears of the gene-edited line and the wild-type self-pollinated ears. The oil content was measured using nuclear magnetic resonance (NMR) spectroscopy. Each ear was weighed three times, and three ears of each material were collected to calculate the oil content.

[0068] Determination of soluble sugar content: Preparation of stock solution: Dissolve 100 mg of glucose, which has been dried to constant weight in an 80°C oven, in 80% 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.

[0069] 1) Sample extraction.

[0070] 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.

[0071] 2) Draw the standard curve.

[0072] 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.

[0073] 3) Measurement.

[0074] 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.

[0075] Determination of amylopectin content: Preparation of stock solution: Weigh 2.0g of potassium iodide, dissolve it in water, then add 0.2g of iodine. After it is completely dissolved, bring the solution to a final volume of 100mL. This final volume solution is the prepared iodine reagent.

[0076] 1) Construction of standard curve.

[0077] Take ten 100ml volumetric flasks and number them. First, take two volumetric flasks and add 0.1g of amylopectin and amylose standards to each. Then, add 1.0ml of ethanol and mix thoroughly. Next, add 9.0mL of 1mol / L sodium hydroxide solution and incubate in a boiling water bath for 10min. Afterward, quickly remove the flasks and cool them in cold water for 5min, then dilute to 100ml. Dilute the amylose and amylopectin standard solutions five times. Take seven 100ml volumetric flasks, add 50ml of ddH₂O to each flask, then add 25.0ml, 24.0ml, 23.0ml, 22.0ml, 21.0ml, 20.0ml, and 19.0ml of the diluted amylopectin standard solution, and 0, 1.0ml, 2.0ml, 3.0ml, 4.0ml, 5.0ml, and 6.0ml of the diluted amylose standard solution, respectively. Take the last 100ml volumetric flask and add 50ml of ddH₂O and 5.0ml of 0.09mol / L sodium hydroxide solution as a blank control. Add 1.0ml of 1mol / L acetic acid solution and 1.0ml of iodine reagent to each volumetric flask, then dilute to 100ml with distilled water. Let stand in the dark for 10 minutes before developing the color. Using ddH2O as a blank control, the absorbance of the sample was measured at a wavelength of 620 nm, and a standard curve was plotted.

[0078] 2) Sample processing and absorbance measurement.

[0079] Add 0.1g of starch sample to a 100ml volumetric flask, followed by 1.0ml of ethanol and 9.0mL of 1mol / L sodium hydroxide solution, and mix well. Then place in a boiling water bath for 10min, followed by immediate cooling in cold water for 5min. Finally, dilute to 100ml with ddH2O. Mix 1ml of ddH2O, 1ml of starch sample solution, 0.5ml of 1mol / L acetic acid solution, and 0.5ml of iodine reagent. After diluting the solution to volume, place in the dark for 10min for color development. Calculate the concentration Y (mg / ml) of amylose in the sample solution based on the standard curve, and then deduce the amylose content in the starch. The sum of the amylose content and amylopectin content in the sample is 100%.

[0080] The results of this embodiment show that, for ZmTHX12 Results of elemental analysis of grains from gene-edited lines, overexpression lines, and wild-type strains. ZmTHXs12 The protein and soluble sugar content in the seeds of the gene-edited lines were significantly increased compared to the wild type; compared to the wild type, the overexpression lines showed increased 100-seed weight, and significantly higher oil and amylopectin content. Figure 5 .

[0081] Based on the above results, we can finally draw the following conclusion: ZmTHX12 Changes in gene expression levels can affect the development of maize kernels. ZmTHX12 Overexpression can increase seed area, increase 100-seed weight, and increase oil content and amylopectin content; ZmTHX12 Gene mutations can increase the protein and soluble sugar content in corn kernels. The results of this invention can lay a theoretical foundation for creating new high-yield, high-protein corn varieties.

[0082] 5. Phylogenetic analysis of ZmTHX12 protein.

[0083] Phylogenetic analysis using the amino acid sequence of ZmTHX12 revealed that the ZmTHX12 protein is conserved in monocotyledonous plants and has a 90% homology protein in maize. Domain prediction showed that this protein contains both a GT1 domain and a PRK14951 domain. (See [link to relevant documentation]). Figure 6 The GT1 domain is a highly conserved functional region in plants, often found in specific transcription factors. Although its topological structure is similar to the DNA-binding domain of Myb protein, its α-helix is ​​longer and it contains conserved sequences capable of binding specific cis-acting elements. It can directly bind to Box II cis-elements in the promoter region of light-responsive genes, and can also bind via Ca2+. 2+ Phosphorylation / dephosphorylation modifications regulate the activity of the PRK14951 domain, which in turn participates in the transcriptional regulation process in plants. The PRK14951 domain is mainly found in the γ and τ subunits of bacterial DNA polymerase III. Both are produced by the same gene through post-translational processing or selective expression, have highly similar sequences, and are highly conserved in Gram-negative and Gram-positive bacteria. Their core functions are related to ATPase activity and protein-protein interactions. The γ subunit acts as the core of the clamp loader, driving the loading and unloading of the sliding clamp onto the DNA through ATPase activity to enhance the binding stability of the polymerase and the template. In addition to retaining similar functions, the τ subunit's C-terminal extension region can also bind to the α subunit of the polymerase core subunit and the helicase DnaB, and act as a replicon linker to coordinate the synergistic synthesis of the leading and lagging strands, ultimately ensuring the efficient replication of bacterial DNA.

[0084] 6. Prediction of the α-sheet protein model of ZmTHX12 protein.

[0085] This invention relates to the amino acid sequence and three-dimensional structural information of the protein encoded by the maize transcript Zm00001eb075250_T003 obtained from the Ensembl Gramene database (accessible at: https: / / ensembl.gramene.org / Zea_mays / Transcript / AFDB?db=core;g=Zm00001eb075250;r=2:23230509-23234898;t=Zm00001eb075250_T003). Analysis shows that this protein contains abundant α-helix structures. Based on the AlphaFold prediction model provided in this database, see [link to relevant documentation]. Figure 7 The 222 amino acid residues, comprising 28.68% of the protein, form an α-helix. The overall folding pattern conforms to the typical structural characteristics of Myb family proteins, and its core α-helix region is the structural basis for its function. The domains appear to be associated with other Myb / SANT-like DN-binding domains, particularly PF10545, which shows the strongest association. This family is significantly amplified in plants and is present in several proteins annotated as transposons. (See...) Figure 8 .

[0086] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0087] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. ZmTHX12 The application of biological products in improving the quality and yield of maize kernels is characterized by, The ZmTHX12 The nucleotide sequence is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.

2.

2. The application as described in claim 1, characterized in that, The biological product is a preparation containing any one or more substances from A to C below: A: The nucleotide sequence shown in SEQ ID NO.1 or its homologous nucleic acid molecules; B: The amino acid sequence shown in SEQ ID NO.2 or its homologous amino acid sequence; C: Substances that can alter the expression levels of A and / or B.

3. The application as described in claim 2, characterized in that, The homologous nucleic acid molecule refers to a nucleic acid molecule that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology with SEQ ID NO.

1. The homologous amino acid sequence refers to an amino acid sequence that has at least 60%, at least 65%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology with SEQ ID NO.

2.

4. The application as described in claim 2, characterized in that, Substances that can alter the expression levels of A and / or B include any one of the following I and II: I: Substances that can increase the expression levels of substances A and / or B; II: Substances that can reduce the expression levels of substances A and / or B.

5. The application as described in claim 4, characterized in that, I is selected from recombinant vectors, expression cassettes, transgenic cell lines, transgenic plant tissues, or recombinant bacteria containing homologous nucleic acid molecules; II is a CRISPR / Cas9 system for knocking out homologous nucleic acid molecules, the CRISPR / Cas9 system comprising a vector for expressing gRNA and a vector for expressing Cas9.

6. The application as described in claim 5, characterized in that, The sequence of the gRNA is shown in SEQ ID NO.

5.

7. The application as described in claim 5, characterized in that, The starting vector for the expression gRNA vector is pBUE411.

8. The application as described in claim 5, characterized in that, The starting vector for the recombinant vector containing homologous nucleic acid molecules is pCambia1300.

9. The application as described in claim 4, characterized in that, I is used to improve the kernel shape, oil content, amylopectin content and yield of corn kernels; II is used to improve the protein and soluble sugar content of corn kernels.

10. The application as described in claim 9, characterized in that, The kernel shape of corn includes kernel length, kernel width, and kernel area.

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