Application of ZmVIM104 Protein in Regulating Maize Kernel Quality Traits

Abstract

The present invention belongs to the field of molecular biology, and relates to the application of ZmVIM104 protein in regulating the quality traits of maize kernels. The amino acid sequence of the ZmVIM104 protein is shown in SEQ ID NO.2, and the nucleotide sequence of the gene encoding the ZmVIM104 protein is shown in SEQ ID NO.1. The present invention discovers that the ZmVIM104 protein can regulate the quality traits of maize kernels. Mutating the ZmVIM104 gene in maize can increase the soluble sugar content of maize kernels and reduce the contents of protein, oil, amino acids and amylose in maize kernels. This discovery can not only provide a scientific basis for analyzing the development mechanism of kernels, but also provide theoretical guidance for the genetic improvement of maize.

Description

Technical Field

[0001] The present invention belongs to the field of molecular biology, and relates to the application of ZmVIM104 protein in regulating the quality traits of maize kernels. Background Art

[0002] Genomic imprinting is an epigenetic phenomenon that occurs in plants and mammals, which depends on the differences in chromatin modifications generated during gametogenesis in both male and female. Genomic imprinting results in the phenomenon that in the seeds of reciprocal crosses in the current generation, only the alleles from one parent are expressed, while the alleles from the other parent are silenced or have weak expression levels. These parent-specifically expressed alleles are defined as imprinted genes. If a gene is a maternally expressed imprinted gene (MEGs), it will only be expressed in the offspring when it comes from the mother. For paternally expressed imprinted genes (PEGs), they will only be expressed in the offspring when they come from the father. Imprinted genes have been shown to be involved in several developmental processes of seeds, such as morphogenesis, dormancy, and postzygotic reproductive isolation. Although hundreds of imprinted genes have been identified in different plants through high-throughput technologies, only a few of their functions have been studied in detail. Therefore, exploring the functions of imprinted genes helps to understand the developmental mechanism of maize kernels, thus providing a theoretical basis for improving maize yield and quality.

[0003] The plant VIM family is a member of a small gene family, encoding proteins containing PHD, RING, and SRA (SET and RING associated) domains, which are commonly present in mammalian proteins involved in chromatin modification, transcription, and cell cycle regulation. Methylcytosine-binding proteins decipher the epigenetic information encoded by DNA methylation and provide a link between DNA methylation, chromatin structure modification, and gene silencing. VIM1 (VARIANT IN METHYLATION 1) encodes an SRA (SET and RING associated) domain methylcytosine-binding protein in Arabidopsis thaliana. Loss of VIM1 function leads to hypomethylation of centromeric DNA and decondensation of interphase centromeric heterochromatin.

[0004] The Arabidopsis ORTH / VIM (ORTHRUS / Variant In Methylation) gene family has six members, which are an orthologous gene group of mammalian UHRF (Ubiquitin-Like Containing, PHD, RINGFINGER). The proteins encoded by ORTH1-ORTH5 possess one PHD domain, two RING domains, and one SRA (SET RING associated) domain. Studies have shown that ORTH proteins have E3 ubiquitin ligase activity in vitro and can mediate DNA methylation. The sixth member, ORL1 / VIM6 (ORTH LIKE-1 / Variant In Methylation 6), only has one RING domain and one SRA domain, and its SRA domain functions to bind methylated DNA. Correct methylation is very important for gene regulation. MET1 (DNA METHYLTRANSFERASE 1) acts on CG methylation of DNA. It has been found that ORTH / VIM can accumulate at corresponding sites by recognizing CG methylation established by MET1 and becomes a key component in the MET1-mediated DNA methylation pathway. On the target genes of vim mutants, active chromatin marks such as H3K4me3 and H3K9 are significantly increased, while repressive chromatin marks such as H3K9me2 and H3K27me3 are decreased. In addition, the deficiency of VIM causes a significant decrease in H3K9me2 in heterochromatic chromocenters. Therefore, VIM proteins play an important role in coordinating the transition of histone modification and DNA methylation status by regulating the activation and repression of histone modifications to silence target genes.

[0005] Grain yield is a multi-factorial trait determined by multiple QTLs and environmental factors. The number of grains and the average grain weight are important components of the yield per plant. Grain weight depends largely on grain size (including grain length, grain width, grain thickness, and the complex trait of grain shape, the length-width ratio). How cereal crops determine the size of their seeds is also a question of particular interest to researchers. Elucidating the genetic mechanisms underlying grain shape and weight regulation is of great significance for the genetic breeding of high-yielding crops. In angiosperms, seed development begins with a double fertilization event, ultimately forming a diploid embryo and a triploid endosperm, which are enclosed by the seed coat formed by maternal tissues. The grain endosperm contains three main cell types: endosperm cells that accumulate starch and store proteins, a transfer layer that absorbs and transports nutrients, and an aleurone layer (AL) that covers the endosperm cells. After pollination, endosperm cells divide rapidly, and around 10 days after pollination, cell differentiation begins. One to several layers of surface cells differentiate into the aleurone layer, and internal cells begin to form plastids to accumulate nutrients such as starch. The starch endosperm cells of mature grains degrade through programmed cell death, spreading from the central region to the peripheral region. During endosperm development, the dynamic process by which the energy of photosynthesis in source organs is accumulated and stored as starch in grains is called grain filling. The grain filling rate determines to some extent the plumpness of grains, ultimately affecting grain weight and yield. In addition to the typical grain structure, there are also some morphological differences in grains of different crops. Notably, maize grains are not covered by glumes, and their grain size mainly depends on the development of the endosperm, especially the basal endosperm transfer layer and AL cells. Grain development involves not only processes of cell proliferation and expansion but also metabolomic processes related to photosynthesis, accumulation, transport, distribution, and storage, as well as many molecular and biological processes, including chromatin modification, transcriptional regulation, translational modification, and protein interactions. With the development of molecular marker and sequencing technologies, more and more genes controlling grain size and weight have been discovered, and these regulatory factors are involved in G protein signal transduction, mitogen-activated protein kinase signal transduction, ubiquitin-proteasome signal transduction, phytohormone signal transduction, or transcriptional regulation. Analyzing the molecular mechanisms of these regulatory factors provides a theoretical basis for new breeding technologies for gramineous crops such as marker-assisted selection and genetic engineering, and is of great significance for the genetic improvement of high-yield and high-quality crops.

[0006] Maize grain is the caryopsis of maize, and its normal development plays a very important role in realizing the value of maize. Maize grain consists of pericarp, testa, embryo, and endosperm. The pericarp and testa are developed from the ovary wall and inner integument respectively, and the two are closely connected and difficult to separate, playing a role in resisting physical or biological damage; the embryo and endosperm are formed by the combination of two sperm cells from the same pollen tube with the egg cell and polar nucleus respectively.

[0007] The embryo of maize develops from the fertilized egg cell and can develop from a single-celled zygote into a micro-plant with five or six leaf primordia and a primary root within 40 - 50 days after pollination. The mature embryo consists of an embryonic axis and a scutellum. The embryonic axis contains the plumule and the radicle, and the scutellum is a large storage organ that is functionally equivalent to the cotyledons in dicotyledonous plants.

[0008] The development of maize endosperm begins with the formation of a triploid endosperm by the fusion of the central cell and the pollen sperm, and then undergoes three main cytological stages: the syncytial stage, the endosperm cellularization stage, and the cell fate specification and proliferation stage, finally forming a mature endosperm structure with completed structural differentiation. The mature endosperm is mainly composed of four types of cells: (1) Basal endosperm transfer layer cells (BETL), located at the base of the endosperm, are connected to the maternal microtubule system through the placenta-chalaza (P-C) and are responsible for transporting nutrients from the maternal tissue to the developing endosperm; (2) The aleurone layer (AL), which forms a single layer of cells on the outermost layer of the endosperm, mainly accumulates some proteins, phospholipids, vitamins, and minerals, and can also provide energy for seed germination by hydrolyzing stored polymers; (3) The starchy endosperm (SE), which is the main storage organ in the endosperm, accumulates a large amount of starch and storage proteins, and the main protein is zein, accounting for about 80% - 90% of the total grain protein; (4) The embryo-surrounding region (ESR), which surrounds the entire young embryo during the early differentiation stage of the endosperm. With the formation of the scutellum of the embryo, one to three layers of endosperm cells adjacent to the scutellum form the endosperm adjacent to scutellum (EAS), and the embryo-surrounding region is mainly attached around the suspensor. This type of cell may be involved in embryo defense and signal transmission between the embryo and the endosperm. The differentiation of different types of cells is crucial for the development of maize grains and the formation of yield.

[0009] A number of mutants related to kernel development have been identified in maize, and their genes have been cloned and functionally characterized. Maize kernel mutants are mainly divided into three categories, namely empty pericarp (emp), defective kernel (dek), and small kernel (smk). Maize dek mutants are a type of maize kernel mutants in which both the embryo and endosperm are damaged during development. A common phenomenon in kernel mutants is that embryo mutations have little effect on the wild-type endosperm, while the loss of function of some genes specifically expressed in the endosperm results in an empty pericarp phenotype, that is, it affects the development of both the endosperm and the embryo. The Emp6 gene encodes an RNA-binding protein, which is essential for the development of both the embryo and endosperm. The emp6 mutation leads to impaired embryo and endosperm development. The genetic regulatory network of maize kernel development is complex, and the functions of a large number of genes related to kernel development have not been deeply studied. Maize kernel development directly determines maize yield. Analyzing the genes during maize kernel development is beneficial to understanding the genetic regulatory mechanism of maize kernel development and providing theoretical support for maize high-yield breeding. Summary of the Invention

[0010] Technical Problem: The inventors previously studied the function of the ZmVIM104 protein and found that the ZmVIM104 gene in mutant maize can reduce kernel weight, kernel area, and embryo area. The results were published in "Xiaomei D, Haishan L, Jiabin Y, et al. The conservation of allelic DNA methylation and its relationship with imprinting in maize [J]. Journal of experimental botany, 2024, 75(5), 1376 - 1389". However, it is unknown whether the ZmVIM104 protein regulates the contents of soluble glycoproteins, oil, amino acids, and amylose in maize kernels.

[0011] Therefore, the first object of the present invention is to provide the application of any one of the following substances 1) - 3) in regulating the quality traits of maize kernels:

[0012] 1) ZmVIM104 protein;

[0013] 2) The gene encoding the ZmVIM104 protein;

[0014] 3) A recombinant vector, expression cassette, transgenic cell line, or recombinant bacterium containing the gene encoding the ZmVIM104 protein;

[0015] The ZmVIM104 protein is as follows (1) or (2):

[0016] (1) A protein consisting of the amino acid sequence shown in SEQ ID NO.2;

[0017] (2) A protein derived from (1) with the same function, which is obtained by substituting and / or deleting and / or adding one or several amino acid residues in the amino acid sequence shown in SEQ ID NO.2.

[0018] The second object of the present invention is to provide the application of a substance that inhibits the expression or activity of the ZmVIM104 protein in regulating the quality traits of maize kernels.

[0019] The third object of the present invention is to provide the application of a substance that inhibits the expression of the gene encoding the ZmVIM104 protein in regulating the quality traits of maize kernels.

[0020] Furthermore, in the regulation of maize kernel traits, it is to increase the soluble sugar and branched-chain starch contents of maize kernels and decrease the protein, oil, amino acid, and amylose contents of maize kernels.

[0021] Furthermore, the substance that inhibits the expression of the gene encoding the ZmVIM104 protein is the CRISPR / Cas9 system, and the CRISPR / Cas9 system is a recombinant vector expressing gRNA and Cas9.

[0022] Furthermore, the target sequence of the gRNA is as shown in SEQ ID NO.3.

[0023] The fourth object of the present invention is to provide a method for obtaining transgenic maize with increased soluble sugar and branched-chain starch contents in kernels and decreased protein, oil, amino acid, and amylose contents in kernels. By inhibiting the expression or activity of the ZmVIM104 protein in maize, transgenic maize is obtained;

[0024] Or by inhibiting the expression of the gene encoding the ZmVIM104 protein in maize, transgenic maize is obtained.

[0025] The fifth object of the present invention is to provide the application of the ZmVIM104 protein or its encoding gene in identifying the quality traits of maize kernels.

[0026] Furthermore, the kernel quality traits include soluble sugar, branched-chain starch content, protein, oil, amino acid, and amylose content.

[0027] Furthermore, quantitatively detect the expression level of the ZmVIM104 protein or its encoding gene in a maize population. If the expression level of the ZmVIM104 protein or its encoding gene in the maize to be tested is lower than the average value of this population, it means that the soluble sugar and branched-chain starch contents of the kernels of the maize to be tested are increased, and the protein, oil, amino acid, and amylose contents of the kernels are decreased.

[0028] The present invention has the following beneficial effects:

[0029] The present invention for the first time discovers that the ZmVIM104 gene in mutant maize can increase the contents of soluble sugar and branched-chain starch in maize kernels, and reduce the contents of protein, oil, amino acids and amylose in maize kernels. This discovery can not only provide a scientific basis for analyzing the development mechanism of kernels, but also provide theoretical guidance for the genetic improvement of maize. Description of the Drawings

[0030] Figure 1 For the identification of the ZmVIM104 gene structure and frameshift mutant lines; A: The target site of ZmVIM104 gene editing; B: The ZmVIM104 gene structure, mutation sites and types.

[0031] Figure 2 For the sequencing peak map of the ZmVIM104 gene mutant.

[0032] Figure 3 For protein structure analysis.

[0033] Figure 4 For the determination of kernel indexes; A: Protein content; B: Oil content; C: Amino acid content; D: Soluble sugar content; E: Amylose content determination; Zmvim104-ref: Wild type KN5585; Zmvim104-C1, Zmvim104-C2: Two frameshift mutant lines of Zmvim104; **: Represents extremely significant at P<0.01. Detailed Embodiments

[0034] The present invention will be described in detail below with reference to the drawings and specific embodiments, but should not be construed as a limitation of the present invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well-known to those skilled in the art. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0035] Example 1: Discovery of the imprinted gene ZmVIM104

[0036] Through transcriptome sequencing analysis experiments, the imprinted gene ZmVIM104 was discovered from the transcriptome data of the reciprocal cross 15-day endosperm of maize inbred line B73 (hereinafter referred to as maize B73) and maize inbred line Mo17 (hereinafter referred to as maize Mo17). In the cDNA of maize B73, the nucleotide sequence of the ZmVIM104 gene is shown in SEQ ID NO.1. The ZmVIM104 gene encodes the protein ZmVIM104. The amino acid sequence of the protein ZmVIM104 is shown in SEQ ID NO.2.

[0037] SEQ ID NO.1:

[0038]

[0039] SEQ ID NO.2:

[0040] MVQQPRINSAIVSAIRAARMSKNTNPAAAGSSAGSHHYIQNVDRPDRAYTTERAKRGGKANASSGQIFVTTAPDHFGPILAEHDPRRNTGVRVGETWEDRLECRQWGAHLPHIAGIAGQSRFGAQSVALSGGYEDDEDHGEWFLYTGSGGRDLSGNKRTNKEQGFDQTFVKMNEALRQSCLRGYPVRVVRSHKEKRSSYAPELGVRYDGIYRIEKCWRKIGVQGTFKVCRYLFVRCDNEPAPWTSDDHGDRPRPLPNIPELKDATDINERDERPSWGYDEKEGRWKWMYEPPTSRKPVRTGLRPKTGKQARKRARSDEMPNDPEKLLNEFSCCICFQVMAEPLTAPCGDSFCKACLLGAYDNQSSVRERSHGGRTLRAQKIVKRCPSCATDISDFLADPQINRDIMDVIESLQRKGDTTTKDDAASYGYGADKAEEFRGGDLQEEADPEMDAEDNSESDTDADASCGRTVVGIKDQEGQQLQPQKRKGGVAIIGTDDGPSKRITASADVEQKGNKSHE。

[0041] Example 2: Construction, Genotype Identification and Phenotype Identification of Gene-Edited Mutant Lines of Imprinted Gene ZmVIM104

[0042] I. Construction of Gene-Edited Mutant Lines of Imprinted Gene ZmVIM104

[0043] 1. Retrieve the complete CDS sequence of the maize ZmVIM104 gene from the MaizeGDB maize genome annotation database, as shown in SEQ ID NO.1, and submit it to Sangon Biotech (Shanghai) Co., Ltd. for gene synthesis;

[0044] 2. Construction of the Crispr / Cas9 vector: As Figure 1As shown in A, first, a 19-bp target sequence (shown in SEQ ID NO.3) was designed for the CDS region near the translation start codon of the ZmVIM104 gene, and the target sequence primers (shown in SEQ ID NO.4 and SEQ ID NO.5) were synthesized. Using 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 genome editing in plants. BMC Plant Biol. 2014 Nov 29;14:327.") as a template for amplification, the PCR product containing the target sequence was purified and recovered. Using 10x BsaI endonuclease and high-concentration T4 ligase, the PCR product and the pBUE411 vector were ligated, transformed into Escherichia coli, and single colonies were selected for identification. The correctly ligated monoclonal colonies were selected, cultured in a shaker, and the plasmids were extracted and stored at -20°C.

[0045] SEQ ID NO.3: 5′-GAATGTTGATAGGCCCGAC-3′ (i.e., positions 120 - 138 from the 5′ end of SEQ ID NO.1).

[0046] SEQ ID NO.4: 5′-TTTGTGCATTTTACTTGGGCTA-3′;

[0047] SEQ ID NO.5: 5′-TGAGCACCCCATTGCCTA-3′.

[0048] 3. Agrobacterium competent cell transformation: The EHA-105 from Weidi Biotechnology was used for the Agrobacterium competent cell transformation experiment. The specific operation steps are as follows:

[0049] (1) Take a tube of Agrobacterium competent cells from the -80°C refrigerator and place it on ice for 15 min to melt.

[0050] (2) Add 1 - 10 μL of the plasmid to be transformed (the total amount does not exceed 100 μg), gently flick the tube wall to mix, and place it on ice for 30 min.

[0051] (3) Place the centrifuge tube containing the competent cells in liquid nitrogen for 5 min, take it out and place it in a 37°C water bath for heat shock for 5 min,

[0052] Take it out and immediately place it on ice for 2 min.

[0053] (4) Add 500 μL of SOC medium, place it in a shaker at 28°C, shake at 180 rpm for 3 - 5 h to activate Agrobacterium.

[0054] (5) Centrifuge for 30 s, aspirate and discard the supernatant, leaving only about 100 μL of liquid. Resuspend the bacterial pellet at the bottom of the tube by pipetting up and down. In a laminar flow hood, aspirate all the bacterial liquid and evenly spread it on an LB solid medium containing the corresponding antibiotic for the plasmid and rifampicin antibiotic. Incubate in an incubator at 28 °C for 1 - 2 days.

[0055] (6) In a laminar flow hood, use a sterilized pipette tip to pick out colonies of appropriate size and transfer them to an LB liquid medium containing the corresponding antibiotic and rifampicin antibiotic. Incubate at 28 °C for 5 - 8 h, then take out for bacterial liquid PCR detection to confirm that the constructed gene editing vector has been correctly transferred into Agrobacterium.

[0056] 4. Agrobacterium - mediated transgenic transformation

[0057] 1) Inoculate the Agrobacterium liquid containing the target plasmid into 50 ml of medium containing rifampicin and kanamycin antibiotics, and shake the culture overnight at 28 °C and 220 rpm.

[0058] 2) Harvest the ears of the donor material KN5585 that have been self - pollinated for 10 - 15 days. After removing the bracts completely, disinfect them in 75% alcohol for 20 min and wash them 3 times in a sterile water beaker.

[0059] 3) Dissect the embryos. In a laminar flow hood sterilized by overnight ultraviolet light, use a scalpel that has been burned and cooled to dig out the young embryos and place them in a 2 - ml centrifuge tube containing the infection bacterial liquid. The size of the young embryos is preferably about 2 mm.

[0060] 4) Transfer the infected embryos to a medium without any screening antibiotics and incubate at 25 °C for 24 hours.

[0061] 5) Invert the plate, transfer the cultured embryos to the medium, with the embryo surface facing down, and space the embryos apart from each other to provide enough space for growth and differentiation. Incubate under the culture condition of 30 °C. Check the contaminated culture dishes during the process. Sterilize the culture dishes contaminated with fungi and transfer the non - contaminated embryos in the culture dishes contaminated with bacteria to new culture dishes. Incubate for about 15 days to allow the embryos to differentiate into callus.

[0062] 6) Select well - growing callus and transfer it to a new culture dish, and incubate at 25 °C to allow it to root and grow into seedlings.

[0063] 7) Transfer the seedlings with callus to a medium containing herbicide for further screening. Select the seedlings with better growth and transfer them to nutrient soil. Grow them in a greenhouse until they flower and set seeds, which are recorded as T0 generation putative transgenic maize.

[0064] II. Genotype identification of the gene - edited mutant lines of the imprinted gene ZmVIM104

[0065] 1. Using the genomic DNA of transgenic maize leaves represented by T0 as a template, PCR amplification was performed with the primer pair 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. The PCR amplification products were respectively subjected to Sanger sequencing. The sequencing results were compared with the target site sequences edited by the CAS9 protein on the ZmVIM104 gene, and plants with heterozygous or homozygous frameshift mutations in the target site region were selected.

[0066] SEQ ID NO.6: 5′-CGTAGACCGATCCCCTACAA-3′;

[0067] SEQ ID NO.7: 5′-ACCCCTCAAGCAGCTTTGTC-3′.

[0068] Table 1 PCR reaction procedure

[0069]

[0070]

[0071] 2. The identified heterozygous mutant strains were respectively self-crossed, and the obtained seeds were T1 generation seeds, and the plants grown from the T1 generation seeds were T1 generation plants; the T1 generation plants were self-crossed, and the obtained seeds were T2 generation seeds, and the plants grown from the T2 generation seeds were T2 generation plants.

[0072] 3. Using the genomic DNA of T1 generation plant leaves as a template, PCR amplification was performed with the primer pair consisting of SEQ ID NO.6 and SEQ ID NO.7, and the corresponding PCR amplification products were obtained. The PCR amplification products were respectively subjected to Sanger sequencing. The sequencing results were compared with the target site sequences edited by the CAS9 protein on the ZmVIM104 gene, and homozygous mutation types were selected.

[0073] 4. The ZmVIM104 genes on the two homologous chromosomes of Zmvim104-C1 had the same mutation, specifically, there were 7-base deletions (129bp - 135bp) in the ZmVIM104 gene on both homologous chromosomes ( Figure 1 B, Figure 2 ), which caused a frameshift and led to the loss of function of the protein ZmVIM104 ( Figure 3 ).

[0074] 5. The ZmVIM104 genes on the two homologous chromosomes of Zmvim104-C2 had the same mutation, specifically, there were 16-base deletions (123bp - 137bp) in the ZmVIM104 gene on both homologous chromosomes (Figure 1 B, Figure 2 ), which causes a frameshift and leads to the loss of function of the protein ZmVIM104( Figure 3 ).

[0075] III. Determination of internal elements in kernels of Zmvim104 homozygous mutant maize

[0076] 1. Plant the materials (maize inbred lines KN5585, Zmvim104-C1, Zmvim104-C2), and self-cross; take the kernels at the mature stage for the determination of starch, protein, oil content, and amino acid content.

[0077] a. Determination of soluble sugar

[0078] Preparation of stock solution: Weigh 100 mg of glucose that has been dried to a constant weight in an 80 °C oven, and dissolve it in 1000 mL of 80% (v / v) ethanol to obtain a glucose standard solution. Weigh 1 g of anthrone and dissolve it in 1000 mL of dilute sulfuric acid solution to obtain anthrone reagent, and place it in a brown bottle for use on the same day.

[0079] 1) Extraction of samples

[0080] Place the test kernels 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, place it in a 10 mL centrifuge tube, add 4 mL of 80% ethanol, place the sample in an 80 °C water bath for 40 min, and stir once every 10 min. Then centrifuge at 5000 g for 5 min and collect the supernatant. Add 0.01 g of activated carbon to the supernatant and perform decolorization treatment at 80 °C for 30 min. Finally, make up the volume to 10 mL and take the filtrate for determination.

[0081] 2) Plot the standard curve

[0082] Prepare glucose solutions with different concentrations of standards, add 5 mL of freshly prepared anthrone reagent on the same day, mix well and place in a boiling water bath for 10 min, then quickly transfer to cold water for cooling for 2 min. Measure the absorbance at a wavelength of 625 nm using a spectrophotometer. Plot the standard curve with glucose content as the abscissa and absorbance as the ordinate, and calculate the standard linear equation.

[0083] 3) Determination

[0084] Take 1 mL of the filtrate from step 1), mix it with 5 mL of anthrone reagent, measure the absorbance in the same way as in step 2), and determine the soluble sugar content according to the standard curve.

[0085] b. Extraction of starch

[0086] The grains were ground into powder and mixed with 0.45% (w / w) sodium metabisulfite in a ratio of 1:2. The filtrate was collected by filtration, centrifuged at 4000 rpm for 10 min in a centrifuge, the supernatant was discarded, sufficient water was added for dissolution, and the process was repeated 3 times. Finally, it was dissolved in ethanol, filtered and dried overnight at 35 °C to obtain pure starch.

[0087] c. Determination of amylose / amylopectin content

[0088] Preparation of stock solution: Weigh 2.0 g of potassium iodide, dissolve it in water, then add 0.2 g of iodine. After complete dissolution, the solution was made up to 100 mL. The solution after volume fixation is the prepared iodine reagent.

[0089] 1) Preparation of standard curve

[0090] Take 10 100-ml volumetric flasks and number them. First, take 2 volumetric flasks and add 0.1 g of amylopectin and amylose standard products to each, then add 1.0 ml of ethanol and mix well. Then add 9.0 mL of 1 mol / L sodium hydroxide solution and water bath in boiling water for 10 min. Then quickly take it out and cool it in cold water for 5 min, and make up to 100 ml. After diluting the amylose and amylopectin standard solutions by 5 times, take another 7 100-ml volumetric flasks, add 50 ml of ddH2O to each flask, and then add 25.0 ml, 24.0 ml, 23.0 ml, 22.0 ml, 21.0 ml, 20.0 ml, 19.0 ml of the diluted amylopectin standard solution, and 0, 1.0 ml, 2.0 ml, 3.0 ml, 4.0 ml, 5.0 ml, 6.0 ml of the amylose standard solution respectively. Take the last 100-ml volumetric flask, add 50 ml of ddH2O and 5.0 ml of 0.09 mol / L sodium hydroxide solution as a blank control. Add 1.0 ml of 1 mol / L acetic acid solution and 1.0 ml of iodine reagent to all volumetric flasks respectively, and then make up to 100 ml with distilled water. Let it stand in the dark for 10 min for color development. Using ddH2O as a blank control, measure the absorbance of the sample at a wavelength of 620 nm and plot the standard curve.

[0091] 2) Treatment of samples and determination of absorbance

[0092] Add 0.1 g of starch sample to a 100-ml volumetric flask, then add 1.0 ml of ethanol and mix well with 9.0 ml of 1 mol / L sodium hydroxide solution. Then place it in a boiling water bath for 10 min, and then quickly take it out and cool it in cold water for 5 min. Finally, make up the volume to 100 ml with ddH2O. Mix 1 ml of ddH2O, 1 ml of starch sample solution, 0.5 ml of 1 mol / L acetic acid solution and 0.5 ml of iodine reagent. After making up the volume of the solution, place it in the dark for color development for 10 min. Calculate the concentration Y (mg / ml) of amylose in the sample solution according to the standard curve, and then deduce the amylose content in the starch.

[0093] d. Determination of protein, oil and amino acids

[0094] Use the Xingling G3100 near-infrared grain analyzer to measure the materials. When measuring, each material should fill the measuring column. Tap the bottom of the measuring column vigorously by hand to make the measuring material fill the measuring column without gaps. Measure three times, and process and analyze the data through Excel software.

[0095] 2. The comparison results of the internal element determination of the Zmvim104 mutant and the wild type are as Figure 4 shown. The contents of protein, oil, amino acids and amylose in the grains of the Zmvim104 mutant are significantly lower than those of the wild type, while the contents of soluble sugar and amylopectin are significantly higher than those of the wild type (amylopectin content + amylose content = 100%, when the amylose content decreases, the amylopectin content increases).

[0096] Although the preferred embodiments of the present invention have been described, those skilled in the art can make additional changes and modifications to these embodiments once they learn the basic creative concepts. Therefore, the appended claims are intended to be construed to include the preferred embodiments as well as all changes and modifications falling within the scope of the present invention.

[0097] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention is also intended to include these modifications and variations.

Claims

1. Application of ZmVIM104 protein in regulating maize kernel quality traits, characterized in that: The amino acid sequence of the ZmVIM104 protein is shown in SEQ ID NO.2, and the quality traits of maize kernels are regulated by inhibiting the expression or activity of the ZmVIM104 protein. The regulation of maize kernel quality traits is to increase the contents of soluble sugar and branched-chain starch in maize kernels and decrease the contents of protein, oil, amino acids and amylose in maize kernels.

2. Use of a substance that inhibits the expression of the ZmVIM104 protein-coding gene described in claim 1 in regulating maize kernel quality traits, characterized in that: The substance for inhibiting the expression of the gene encoding the ZmVIM104 protein is the CRISPR / Cas9 system. The CRISPR / Cas9 system is a recombinant vector expressing gRNA and Cas9, and the target sequence of the gRNA is shown in SEQ ID NO.3; the regulation of maize kernel quality traits is to increase the contents of soluble sugar and branched-chain starch in maize kernels and decrease the contents of protein, oil, amino acids and amylose in maize kernels.

3. A method for obtaining a transgenic maize with increased soluble sugar and branched-chain starch contents in grains and decreased protein, oil, amino acid, and amylose contents, characterized in that: Transgenic maize is obtained by inhibiting the expression or activity of the ZmVIM104 protein in maize; or transgenic maize is obtained by inhibiting the expression of the gene encoding the ZmVIM104 protein in maize; The ZmVIM104 protein is the ZmVIM104 protein described in claim 1.

Images