Micropeptide and use thereof

Abstract

The present application relates to a kind of micropeptide and its application, belong to the field of genetic engineering.The present application provides a kind of micropeptide microRPG1 unique in maize plant.The present application finds that the preparation prepared using microRPG1 has multiple effects such as regulating plant grain moisture content, delaying fruit ripening, delaying aging, prolonging life span, reducing active oxygen generation, etc., and can be developed into cosmetic raw materials, medicines, health products, plant nutrient solutions and other products, for use in the fields of improving plant grain moisture content, delaying fruit ripening, antioxidant, delaying aging, etc.Further, the micropeptide in corn and the encoding gene are inhibited, which can accelerate the dehydration of corn kernels, and have important application value.

Description

Technical Field

[0001] This invention relates to a micropeptide and its applications, belonging to the field of genetic engineering. Background Technology

[0002] Micropeptides are peptides encoded by small open reading frames (sORFs) of no more than 100 codons. Increasing research indicates that micropeptides have significant applications in areas such as organismal developmental regulation and targeted disease therapy.

[0003] With the increasing aging of the global population, interest in "proactive health and delaying aging" is growing, primarily based on scientific evidence that a series of basic mechanisms targeting aging can delay or exacerbate various age-related chronic or non-communicable diseases. Therefore, cellular senescence has garnered significant attention as a potential target for preventing or treating various age-related diseases and improving healthy lifespan.

[0004] Cellular senescence refers to a relatively stable and usually irreversible state of cell cycle arrest in eukaryotic cells. In this state, proliferating cells develop tolerance to growth-promoting stimuli, typically caused by stress signals such as DNA damage. Replicative senescence refers to the cessation of continuous cell division in normal cells after approximately 30-50 divisions. Replicative senescence is essentially induced by the progressive shortening of telomeres. During each round of DNA replication, telomeres gradually shorten, eventually reaching a critical length that prevents further replication, thereby halting cell division. Shorter uncapped telomeres elicit a DNA damage response, directly triggering senescence.

[0005] Delaying aging has significant implications for improving crop yields and stress resistance, treating human diseases, and increasing human well-being. Therefore, preparations, drugs, health products, foods, and plant nutrient solutions with anti-aging effects all possess enormous commercial value.

[0006] Grain moisture content is a key factor affecting the quality of mechanized corn harvesting, safe storage, and economic benefits. The moisture content of the grains at harvest has a significant impact on corn harvesting, drying, storage, transportation, and processing. Excessive moisture content often causes economic losses for corn growers and operators, reduces economic benefits, and easily leads to grain mold, affecting corn quality. Furthermore, mechanized grain harvesting has become one of the main factors limiting corn production in my country, and the most critical aspect of mechanized grain harvesting is ensuring that the corn grain moisture content at harvest does not reach the standard of ≤25% for mechanized grain harvesting (Wang Z, Wang X, Zhang L, Liu X, Di H, Li T, Jin X. QTL underlying field grain drying rate after physiological maturity in maize ( Zea Mays L.)[J]. Euphytica, 2012, 185(3):521-528.). Therefore, the breeding of maize varieties with low grain moisture content at harvest is very important. In addition, low grain moisture content can shorten the growth cycle of maize, which is of great significance for pre-frost harvesting in high-latitude areas of my country and for not affecting wheat planting in the Huang-Huai-Hai region. Summary of the Invention

[0007] This invention identifies a gene, ZmRPG, that controls the moisture content and dehydration rate of maize kernels using various genetic and molecular biological methods. It also identifies the micropeptide microRPG1 encoded by the ZmRPG gene and similar micropeptides found in teosinte. Using the ZmRPG gene or the microRPG1 micropeptide, maize varieties with different moisture contents and dehydration rates can be bred as needed. More importantly, microRPG1 also has multiple functions, including regulating kernel moisture content, delaying fruit ripening, anti-oxidation, and delaying aging. It can be developed into cosmetic raw materials, pharmaceuticals, health products, plant nutrient solutions, and other products for improving plant kernel moisture content, delaying fruit ripening, anti-oxidation, and delaying aging.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] The present invention provides a micropeptide, characterized in that the amino acid sequence of the micropeptide is as shown in any one of SEQ ID NO. 4 or SEQ ID NO. 11 to SEQ ID NO. 18.

[0010] The present invention also provides a formulation characterized in that the micropeptide is dissolved in a specific reagent at a concentration of 1 μM to 50 μM; in some embodiments, the above-mentioned reagent includes: water, bacterial solution, cell culture medium, plant culture medium, and physiological saline.

[0011] The present invention also provides uses of the above-mentioned micropeptides or formulations, characterized in that the uses include any of the following:

[0012] 1) Regulate the water content of plant seeds; 2) Delay fruit ripening; 3) Delay senescence; 4) Extend lifespan; 5) Reduce the generation of reactive oxygen species.

[0013] The present invention also provides the application of nucleic acid molecules in improving the moisture content or dehydration rate of corn kernels, characterized in that the nucleotide sequence or reverse complementary sequence of the nucleic acid is shown as any one of SEQ ID NO. 1 to SEQ ID NO. 3.

[0014] The present invention also provides a method for reducing the moisture content of corn kernels or increasing the dehydration rate, characterized in that: the expression and / or activity of the micropeptide of the sequence shown in SEQ ID NO. 4 or the nucleic acid molecule of any one of the sequences shown in SEQ ID NO. 1 to SEQ ID NO. 3 in corn are inhibited, and plants with reduced moisture content of corn kernels or increased dehydration rate are selected;

[0015] In some embodiments, the methods for inhibiting the expression and / or activity of protein or nucleic acid molecules include gene editing or RNA interference;

[0016] In some implementations, the target DNA sequence for gene editing is shown in SEQ ID NO. 5.

[0017] This invention also provides a kit for reducing the moisture content of corn kernels or increasing the dehydration rate, characterized in that it comprises any one of the following:

[0018] (1) An RNA molecule capable of recognizing the above target sequence; in some embodiments, the sequence of the RNA molecule is shown in SEQ ID NO. 6;

[0019] (2) The DNA molecule encoding the RNA described in (1);

[0020] (3) A vector for expressing the RNA described in (1).

[0021] The present invention also provides a mutant gene, characterized in that: the nucleic acid sequence of the mutant gene is shown in any one of SEQ ID NO. 7 to SEQ ID NO. 9.

[0022] The present invention also provides a method for increasing the moisture content of corn kernels or reducing the dehydration rate, characterized in that the expression and / or activity of a micropeptide with the sequence shown in SEQ ID NO. 4 or a nucleic acid molecule with any one of the sequences shown in SEQ ID NO. 1 to SEQ ID NO. 3 is increased in the corn material to be improved, and plants with increased corn kernel moisture content or reduced dehydration rate are selected;

[0023] In some implementations, the method of increasing expression is to drive the expression of nucleic acid molecules using a highly active promoter;

[0024] In some implementations, the highly active promoter is the maize ubiquitin promoter;

[0025] In some implementations, the corn ubiquitin promoter sequence is shown in SEQ ID NO. 10.

[0026] The present invention also provides the application of the above-mentioned methods, kits, mutant genes, and methods in improving the traits of corn kernel moisture content or dehydration rate.

[0027] Compared with existing technologies, the beneficial effects of this invention are as follows: This invention provides a previously unreported ZmRPG gene and its encoded micropeptide microRPG1. This micropeptide has the function of controlling the water content or dehydration rate of maize kernels, and can be used to screen for maize kernel water content or dehydration rate traits, thereby breeding maize varieties with low water content and rapid dehydration. In addition, the microRPG1 micropeptide also has important application value in anti-aging in plants and animals. Attached Figure Description

[0028] Figure 1 qKDR1 Fine mapping and cloning: A: Initial mapping results of QTL linkage analysis (curves of different colors represent different degrees of AUDDC index in BLUP); B: Genotypes of HIF material segments; C: qKDR1 Fine-grained positioning, phenotype is the AUDDC value between two moisture measurements; D: NIL material plant and ear.

[0029] Figure 2 Distribution of 17 differentially expressed genes in 4 NIL populations (R1~R4). A: Downregulated genes; B: Upregulated genes. The numbers in the circles represent the number of overlapping genes.

[0030] Figure 3 qKDR1 Regulation of ZmRPG gene expression. A: Genotypic diagram of the four NIL populations; B: Expression level of ZmRPG gene in the four NIL populations.

[0031] Figure 4The distribution of the 12 open reading frames (ORFs) of the ZmRPG gene. Among them, ORF1 encodes the micropeptide microRPG1.

[0032] Figure 5 Overexpression of ZmRPG slows down the dehydration rate of maize kernels. A: Expression cassette structure of ZmRPG overexpression; B: Expression level of ZmRPG in two transgenic overexpression lines; C: Kernel dehydration rate of ZmRPG overexpression lines at the Hainan experimental site in 2021 (expressed as AUDDC value between two moisture measurements); D: Kernel dehydration rate of ZmRPG overexpression lines at the Jilin experimental site in 2021 (expressed as AUDDC value between two moisture measurements).

[0033] Figure 6 Overexpression of microRPG1 slows down the dehydration rate of maize kernels. A: Expression cassette structure of microRPG1 overexpression; B: Expression level of microRPG1 in two transgenic overexpression lines; C: Results from the Hainan experimental site in 2021; D: Results from the Beijing experimental site in 2022; E: Results from the Jilin experimental site in 2022. The phenotype is the AUDDC value between two moisture measurements.

[0034] Figure 7 Knocking out microRPG1 accelerates the dehydration rate of corn kernels. A: Results from the Beijing test site in 2020; B: Results from the Hainan test site in 2021; C: Results from the Beijing test site in 2022; D: Results from the Jilin test site in 2022. Data are expressed as AUDDC values ​​between two moisture measurements.

[0035] Figure 8 microRPG1 peptides delay the maturation of Arabidopsis thaliana siliques. A: Plant phenotype after microRPG1 treatment; B: Silique phenotype after microRPG1 treatment; C: Seed phenotype after microRPG1 treatment; D: Phenotype at the beginning of silique maturation; E: Phenotype at 50% silique maturity; F: Seed water content; G: Flowering stage performance.

[0036] Figure 9 Effects of different concentrations of microRPG1 peptide exogenous application on silique maturation. A: Phenotype of the treatment at the beginning of silique maturation; B: Phenotype of the treatment at 50% silique maturity. n represents the number of plants.

[0037] Figure 10 microRPG1 peptides can be absorbed by Arabidopsis thaliana roots. Images under FAM-ORF1 fluorescence; Bright-field: images under bright-field conditions; Merge: overlay of fluorescence and bright-field images.

[0038] Figure 11microRPG1 peptides can enhance human cell viability (left) and reduce the production of reactive oxygen species at the cellular level (right).

[0039] Figure 12 Nematode intestinal particles after feeding ORF1 (N2 / OP50+ORF1) and control (N2 / OP50). Day 15 / 18 / 21 / 24 indicates the number of days after feeding.

[0040] Figure 13 Statistical analysis results of the survival days of nematodes after feeding ORF1 (N2+ORF1) and control (N2). Detailed Implementation

[0041] The following definitions and methods are provided to better define this application and to guide those skilled in the art in its practice. Unless otherwise stated, the terms are to be understood in accordance with their conventional usage by those skilled in the art. All patent literature, academic papers, industry standards, and other publicly available publications cited herein are incorporated herein by reference in their entirety.

[0042] As used herein, “maize” means any maize plant and includes all plant varieties that can be bred with maize, including the whole plant, plant cells, plant organs, plant protoplasts, plant cell tissue cultures from which the plant can regenerate, plant callus, and complete plant cells in a plant or plant part, such as embryo, pollen, ovule, seed, leaf, flower, branch, fruit, stem, root, root tip, anther, etc. Unless otherwise indicated, nucleic acids are written from left to right in a 5' to 3' direction; amino acid sequences are written from left to right in the amino to carboxyl direction. Amino acids may be represented herein by their commonly known three-letter symbols or by the single-letter symbols recommended by the IUPAC-IUB Committee on Biochemistry Nomenclature. Similarly, nucleotides may be represented by commonly accepted single-letter codes. Numerical ranges include numbers that define the range. As used herein, “nucleic acid” includes deoxyribonucleotides or ribonucleotide polymers in single-stranded or double-stranded form, and, unless otherwise limited, includes known analogs (e.g., peptide nucleic acids) that have the basic properties of natural nucleotides and hybridize with single-stranded nucleic acids in a manner similar to that of naturally occurring nucleotides. As used herein, the term “encoding” or “encoded” in the context of a particular nucleic acid means that the nucleic acid contains the essential information to guide the translation of that nucleotide sequence into a particular protein. Codons are used to represent the information encoding the protein. As used herein, “full-length sequence” referring to a particular polynucleotide or the protein it encodes means the entire nucleic acid sequence or the entire amino acid sequence having a natural (non-synthetic) endogenous sequence. Full-length polynucleotides encode the full-length, catalytically active form of that particular protein. The terms “polypeptide,” “polypeptide,” and “protein” are used interchangeably herein to refer to polymers of amino acid residues. This term is used for amino acid polymers in which one or more amino acid residues are artificial chemical analogs of the corresponding naturally occurring amino acids. This term is also used for naturally occurring amino acid polymers. The terms “residue” or “amino acid residue” or “amino acid” are used interchangeably in this document to refer to an amino acid incorporated into a protein, polypeptide, or peptide (collectively, “protein”). Amino acids can be naturally occurring amino acids, and unless otherwise limited, may include known analogs of natural amino acids that can function in a similar manner to naturally occurring amino acids.

[0043] The term "trait" refers to the physiological, morphological, biochemical, or physical characteristics of a plant or a particular plant material or cell. In some cases, this trait is visible to the human eye, such as seed or plant size, or can be measured by biochemical techniques, such as detecting the protein, starch, or oil content of seeds or leaves, or by observing metabolic or physiological processes, such as by measuring tolerance to water deprivation or specific salt, sugar, or nitrogen concentrations, or by observing the expression levels of one or more genes, or by agronomic observations such as tolerance to osmotic stress or yield.

[0044] "Plant" includes indexes for whole plants, plant organs, plant tissues, seeds, and plant cells, as well as their offspring. Plant cells include, but are not limited to, cells from seeds, suspension cultures, plumules, meristematic regions, callus, leaves, roots, seedlings, gametophytes, sporophytes, pollen, and microspores. "Offspring" includes any subsequent generations of a plant.

[0045] In this application, the terms "comprising," "including," or variations thereof should be understood to include other elements, numbers, or steps besides those described. "Test plant" or "test plant cell" refers to a plant or plant cell in which genetic modification has taken effect, or a progeny cell of such a modified plant or cell containing the modification. "Control," "control plant," or "control plant cell" provides a reference point for measuring phenotypic changes in the test plant or plant cell.

[0046] Negative or control plants may include, for example: (a) wild-type plants or cells, i.e., plants or cells with the same genotype as the genetically modified starting material, the genetic modification producing the test plants or cells; (b) plants or plant cells with the same genotype as the starting material but transformed with an empty construct (i.e., a construct with no known effect on the target trait, such as a construct containing the target gene); (c) plants or plant cells that are non-transformed isomers of the test plants or plant cells; (d) plants or plant cells that are genetically identical to the test plants or plant cells but not exposed to conditions or stimuli that would induce the expression of the target gene; or (e) the test plants or plant cells themselves, which are under conditions where the target gene is not expressed.

[0047] Those skilled in the art will readily recognize that advances in molecular biology, such as site-specific and random mutagenesis, polymerase chain reaction methods, and protein engineering techniques, have provided a wide range of appropriate tools and procedures for modifying or engineering the amino acid sequences and potential gene sequences of proteins of interest in agriculture.

[0048] In some embodiments, the nucleotide sequence of this application may be modified to perform conserved amino acid substitutions. Principles and examples of conserved amino acid substitutions are further described below. In some embodiments, the nucleotide sequence of this application may be substituted without altering the amino acid sequence according to disclosed monocotyledonous codon preferences; for example, a codon encoding the same amino acid sequence may be substituted with a codon preferred by monocotyledons without changing the amino acid sequence encoded by the nucleotide sequence. In some embodiments, a portion of the nucleotide sequence in this application may be substituted with a different codon encoding the same amino acid sequence, thereby changing the nucleotide sequence without altering the encoded amino acid sequence. Conserved variants include those sequences that encode an amino acid sequence of one of the proteins of the embodiments due to genetic codon degeneracy. In some embodiments, a portion of the nucleotide sequence in this application may be substituted according to a codon preferred by monocotyledons. Those skilled in the art will recognize that amino acid additions and / or substitutions are generally based on the relative similarity of amino acid side-chain substituents, such as the hydrophobicity, charge, size, etc., of the substituents. Exemplary amino acid substituents having the various properties considered above are well known to those skilled in the art and include arginine and lysine; glutamic acid and aspartic acid; serine and threonine; glutamine and asparagine; and valine, leucine, and isoleucine. Guidance on appropriate amino acid substitutions that do not affect the biological activity of the target protein can be found in the model of Dayhoff et al. (1978), *Atlas of Protein Sequence and Structure* (Natl. Biomed. Res. Found., Washington, D. C.) (incorporated herein by reference). Conserved substitutions, such as replacing one amino acid with another amino acid having similar properties, can be performed. Identification of sequence consistency includes hybridization techniques. For example, a known nucleotide sequence, in whole or in part, can be used as a probe for selective hybridization with other corresponding nucleotide sequences present in cloned genomic DNA fragments or cDNA fragment groups (i.e., genomic libraries or cDNA libraries) from a selected organism. The hybridization probe may be a genomic DNA fragment, cDNA fragment, RNA fragment, or other oligonucleotide, and may be labeled with a detectable group such as 32P or other detectable markers. Thus, for example, hybridization probes can be prepared by labeling synthetic oligonucleotides based on sequences from the embodiment. Methods for preparing hybridization probes and constructing cDNA and genomic libraries are generally known in the art. Hybridization of the sequences can be performed under stringent conditions. As used herein, the terms "stringent conditions" or "stringent hybridization conditions" refer to conditions under which the probe will hybridize with its target sequence to a detectable extent (e.g., at least 2, 5, or 10 times the background) relative to hybridization with other sequences.Harshness conditions are sequence-dependent and vary across different environments. By controlling hybridization harshness and / or washing conditions, target sequences 100% complementary to the probe can be identified (homologous probe method). Alternatively, harshness conditions can be adjusted to allow for some sequence mismatches in order to detect lower similarities (heterologous probe method). Typically, probe lengths are less than about 1000 or 500 nucleotides. Typically, harshness conditions are those where the salt concentration is less than about 1.5 M Na ions at pH 7.0 to 8.3, typically about 0.01 M to 1.0 M Na ion concentration (or other salts), and the temperature conditions are: at least about 30°C for short probes (e.g., 10 to 50 nucleotides) and at least about 60°C for long probes (e.g., greater than 50 nucleotides). Harshness conditions can also be achieved by adding a destabilizing agent such as formamide. Exemplary low-tightness conditions include hybridization at 37°C using 30% to 35% formamide buffer, 1 M NaCl, and 1% SDS (sodium dodecyl sulfate), followed by washing at 50°C to 55°C in 1 to 2× SSC (20× SSC = 3.0 M NaCl / 0.3 M trisodium citrate). Exemplary medium-tightness conditions include hybridization at 37°C using 40% to 45% formamide, 1.0 M NaCl, and 1% SDS, followed by washing at 55°C to 60°C in 0.5× to 1× SSC. Exemplary high-tightness conditions include hybridization at 37°C using 50% formamide, 1 M NaCl, and 1% SDS, followed by a final wash at 60°C to 65°C in 0.1× SSC for at least about 20 minutes. Optionally, the wash buffer may contain about 0.1% to about 1% SDS. Hybridization duration is typically less than about 24 hours, typically from about 4 hours to about 12 hours. Specificity typically depends on post-hybridization washing, with key factors being the ionic strength and temperature of the final washing solution. The Tm (thermodynamic melting point) of DNA-DNA hybrids can be approximated by the formula from Meinkoth and Wahl (1984) Anal. Biochem. 138: 267-284: Tm = 81.5℃ + 16.6(logM) + 0.41(%GC) - 0.61(%formamide) - 500 / L; where M is the molar concentration of monovalent cations, %GC is the percentage of guanosine and cytosine nucleotides in the DNA, "formamide%" is the percentage of formamide in the hybridization solution, and L is the base pair length of the hybrid. Tm is the temperature at which 50% of the complementary target sequence hybridizes with a perfectly matched probe (at a given ionic strength and pH). Washing is typically performed at least until equilibration is reached and a low hybridization background level is achieved, such as for 2 hours, 1 hour, or 30 minutes. Each 1% mispairing should lower Tm by approximately 1°C; therefore, Tm, hybridization, and / or washing conditions can be adjusted to hybridize with the desired sequence of consistency.For example, if a sequence with ≥90% homology is required, the Tm can be lowered by 10°C. Typically, stringency conditions are chosen to be approximately 5°C lower than the Tm of the specific sequence and its complementary sequence at the defined ionic strength and pH. However, under very stringent conditions, hybridization and / or washing can be performed at 4°C lower than the stated Tm; under moderately stringent conditions, hybridization and / or washing can be performed at 6°C lower than the stated Tm; and under low stringency conditions, hybridization and / or washing can be performed at 11°C lower than the stated Tm.

[0049] In some embodiments, a fragment of a nucleotide sequence and the amino acid sequence it encodes is also included. As used herein, the term "fragment" refers to a portion of the nucleotide sequence of a polynucleotide of an embodiment or a portion of the amino acid sequence of a polypeptide. A fragment of the nucleotide sequence may encode a protein fragment that retains the biological activity of the native or corresponding full-length protein and thus has protein activity. Mutant proteins include biologically active fragments of native proteins containing consecutive amino acid residues that retain the biological activity of the native protein. Some embodiments also include transformed plant cells or transgenic plants containing a nucleotide sequence of at least one embodiment. In some embodiments, plants are transformed using an expression vector containing a nucleotide sequence of at least one embodiment and a promoter operatively linked thereto that drives expression in plant cells. Transformed plant cells and transgenic plants represent plant cells or plants whose genome contains a heteropolynucleotide. Generally, the heteropolynucleotide is stably integrated into the genome of the transformed plant cell or transgenic plant to pass the polynucleotide to offspring. The heteropolynucleotide may be integrated into the genome alone or as part of an expression vector. In some embodiments, the plants involved in this application include plant cells, plant protoplasts, plant cell tissue cultures capable of regenerating plants, plant callus, plant masses, and plant cells that are whole plants or parts of plants, such as embryos, pollen, ovules, seeds, leaves, flowers, branches, fruits, kernels, ears, rachis, husks, straw, roots, root tips, anthers, etc. This application also includes plant cells, protoplasts, tissues, callus, embryos, flowers, stems, fruits, leaves, and roots derived from transgenic plants of this application or their progeny, and thus at least partially containing the nucleotide sequences of this application.

[0050] In the context of nucleic acid amplification, the term "amplification" refers to any process in which additional copies of a selected nucleic acid (or its transcribed form) are produced. Common amplification methods include replication methods based on various polymerases, including polymerase chain reaction (PCR), ligase-mediated methods such as ligase chain reaction (LCR), and amplification methods based on RNA polymerases (e.g., via transcription).

[0051] An allele is “associated” with a trait when it is linked to the trait, and when the presence of an allele is an indicator that the desired trait or the form of the trait will occur in a plant containing the allele.

[0052] As used in this article, the term "quantitative trait locus" or "QTL" refers to a polymorphic locus that has at least one allele associated with differential expression of a phenotypic trait in at least one genetic context (e.g., in at least one breeding population or offspring). QTLs can function through single-gene or multi-gene mechanisms.

[0053] The term "QTL mapping" used in this article refers to the method of locating QTLs on a genetic map using methods similar to single-gene mapping, determining the distance between the QTL and the genetic marker (expressed as recombination rate). Depending on the number of markers, it can be divided into single-marker, double-marker, and multi-marker methods. Based on the statistical analysis methods, it can be divided into variance and mean analysis, regression and correlation analysis, moment estimation, and maximum likelihood estimation, etc. Based on the number of marker intervals, it can be divided into zero-interval mapping, single-interval mapping, and multi-interval mapping. In addition, there are comprehensive analysis methods that combine different methods, such as QTL composite interval mapping (CIM), multi-interval mapping (MIM), multiple QTL mapping, and multi-trait mapping (MTM).

[0054] The term "molecular marker" as used in this article refers to a specific DNA segment that reflects a certain difference in the genome of an individual or population.

[0055] The term "major gene" used in this article refers to a gene that determines a trait by a single gene. The term "minor gene" refers to several non-allelic genes that each has only a partial influence on the phenotype of the same trait; such genes are called additive genes or polygenes. In additive genes, each gene has only a small phenotypic effect, hence the name minor gene.

[0056] The term "inbred line" used in this article refers to a line obtained by self-pollinating under artificially controlled self-pollination for several generations, continuously eliminating undesirable rows of ears, and selecting individual plants with better agronomic traits for self-pollination, thereby obtaining a line with more uniform agronomic traits and a simpler genetic basis.

[0057] The term "backcross" as used in this article refers to the method of hybridizing the F1 generation with either of the two parents.

[0058] As used herein, the term "hybridization" or "hybrid" refers to the fusion of gametes (e.g., cells, seeds, or plants) that produce offspring through pollination. This term includes sexual hybridization (one plant being pollinated by another) and self-pollination (self-pollination, such as when pollen and ovules come from the same plant). The term "hybridization" refers to the gamete fusion that produces offspring through pollination.

[0059] The term "backcross" as used in this article refers to the process in which the hybrid offspring are repeatedly backcrossed with one of the parents. In a backcross scheme, the "donor" parent refers to the parental plant that possesses the desired gene or locus to be infiltrated. The "recipient" parent (used once or multiple times) or "recurrent" parent (used twice or multiple times) refers to the parental plant into which the gene or locus is infiltrated. The initial hybridization produces the F1 generation; then, the term "BC1" refers to the second use of the recurrent parent, "BC2" refers to the third use of the recurrent parent, and so on.

[0060] As used herein, the term "tightly linked" refers to a recombination frequency between two linked loci of equal to or less than about 10% (i.e., a segregation frequency of no more than 10 cM on a genome map). In other words, tightly linked loci co-segregate at least 90% of the time. Marker loci are particularly useful in this invention when they show a significant probability of co-segregation (linkage) with a desired trait (e.g., pathogen resistance). Tightly linked loci, such as marker loci and second loci, may show an intralocular recombination frequency of 10% or less, preferably about 9% or less, more preferably about 8% or less, more preferably about 7% or less, more preferably about 6% or less, more preferably about 5% or less, more preferably about 4% or less, more preferably about 3% or less, more preferably about 2% or less. In a highly preferred embodiment, the associated loci show a recombination frequency of about 1% or less, for example, about 0.75% or less, more preferably about 0.5% or less, more preferably about 0.25% or less. Two loci located on the same chromosome, and whose distance between them such that the recombination frequency between the two loci is less than 10% (e.g., approximately 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.75%, 0.5%, 0.25%, or lower), are also referred to as "close to each other." In some cases, two different markers can have the same genomic map coordinates. In that case, the two markers are close enough that the recombination frequency between them is so low as to be undetectable.

[0061] Centiliter (“cM”) is a unit of measurement for recombination frequency. 1 cM is equal to the 1% probability that a marker at one locus will separate from a marker at a second locus after a single-generation cross.

[0062] A "favorable allele" is an allele at a specific locus that confers or contributes to an agronomically desired phenotype, such as increased kernel water content in maize, and allows for the identification of plants with the agronomically desired phenotype. A marked "favorable" allele is a marker allele that cosegregates with the favorable phenotype.

[0063] A "gene map" is a description of gene linkages between loci on one or more chromosomes in a given species, typically presented as a graph or table. For each gene map, the distance between loci is measured by the frequency of recombination between them, and recombination between loci can be detected using various markers. A gene map is the product of the mapping population, the types of markers used, and the polymorphic potential of each marker across different populations. The order and genetic distance between loci in one gene map may differ from those in another. However, using a general frame of common markers allows for the association of information between one map and another. Those skilled in the art can use the frame of common markers to identify marker locations and loci of interest on individual gene maps.

[0064] "Genographic location" is the position on the gene map relative to the surrounding genetic markers on the same linkage group, where the specified marker can be found in a given population.

[0065] "Gene mapping" is a method for defining linkage relationships at gene loci, which is performed using genetic markers, marker segregation, and standard genetic principles of recombination frequency.

[0066] "Genetic recombination frequency" is the frequency of exchange events (recombination) between two loci. Recombination frequency can be observed after marker and / or segregation of traits following meiosis.

[0067] The term "genotype" is the genetic makeup of an individual (or group of individuals) at one or more loci, as opposed to an observable trait (phenotype). A genotype is defined by the alleles at one or more known loci that the individual has inherited from their parents. The term genotype can be used to refer to an individual's genetic makeup at a single locus, at multiple loci, or more generally, to refer to the genetic makeup of all the genes in an individual's genome.

[0068] "Germium" refers to the genetic material of an individual (e.g., a plant), a group of individuals (e.g., a plant strain, variety, or family), or a clone derived from or obtained from a strain, variety, species, or culture. Germium can be a part of an organism or cell, or can be isolated from an organism or cell. Germium typically provides genetic material and specific molecular structure that provides the physical basis for some or all of the heritable traits of an organism or cell culture. As used herein, germplasm includes cells, seeds, or tissues from which new plants can grow, or plant parts such as leaves, stems, pollen, or cells that can be cultured into a whole plant.

[0069] A “marker” is a nucleotide sequence or its encoded product (e.g., a protein) used as a reference point. For markers used to detect recombination, they need to detect differences or polymorphisms within the monitored population. For molecular markers, this means that differences at the DNA level are due to differences in multiple nucleotide sequences (e.g., SSRs, RFLPs, FLPs, and SNPs). Genomic variability can originate from any source, such as the presence and sequence of insertions, deletions, duplications, repetitive elements, point mutations, recombination events, or transposons. Molecular markers can be derived from the genome or expressed nucleic acids (e.g., ESTs) and can also refer to nucleic acids used as probes or primer pairs that can amplify sequence fragments using PCR-based methods.

[0070] Markers corresponding to genetic polymorphisms among population members can be detected using methods established in the art. These methods include, for example, DNA sequencing, PCR-based sequence-specific amplification methods, restriction fragment length polymorphism detection (RFLP), isoenzyme labeling detection, polynucleotide polymorphism detection (ASH) via allele-specific hybridization, amplified variable sequence detection of plant genomes, autonomous sequence replication detection, simple repeat sequence detection (SSR), single nucleotide polymorphism detection (SNP), or amplified fragment length polymorphism detection (AFLP). Established methods are also known for detecting expressed sequence tags (ESTs) and SSR markers derived from EST sequences, as well as randomly amplified polymorphic DNA (RAPD).

[0071] A “marker allele” or “marker locus allele” can refer to one of several polymorphic nucleotide sequences located at a marker locus in a population, which is polymorphic with respect to the marker locus.

[0072] A “labeled probe” is a nucleic acid sequence or molecule that can be used to identify the presence or absence of a marker locus by nucleic acid hybridization, such as a nucleic acid molecular probe complementary to a marker locus sequence. A labeled probe containing 30 or more adjacent nucleotides (“all or part” of the marker locus sequence) can be used for nucleic acid hybridization. Alternatively, in some respects, a molecular probe refers to any type of probe capable of distinguishing (i.e., genotyping) specific alleles present at a marker locus.

[0073] As described above, when identifying linked loci, the term "molecular marker" can be used to refer to a genetic marker, or its encoded product (e.g., a protein) used as a reference point. Markers can be derived from genomic nucleotide sequences or expressed nucleotide sequences (e.g., from spliced ​​RNA, cDNA, etc.), or from encoded polypeptides. The term also refers to nucleic acid sequences complementary to or flanked by marker sequences, such as nucleic acids used as probes or primer pairs capable of amplifying marker sequences. A "molecular marker probe" is a nucleic acid sequence or molecule that can be used to identify the presence or absence of a marker locus, such as a nucleic acid probe complementary to a marker locus sequence. Alternatively, in some respects, a molecular probe refers to any type of probe capable of distinguishing (i.e., genotype) specific alleles present at a marker locus. Nucleic acids are "complementary" when they hybridize specifically in solution, for example, according to the Watson-Crick base pairing principle. Some markers described herein are also called hybridization markers when located in insertion / deletion regions, such as the non-collinear regions described herein. This is because insertion regions are polymorphisms concerning the absence of insertions. Therefore, the marker only needs to indicate the presence or absence of the insertion / deletion region. Any suitable marker detection technique can be used to identify such hybridization markers, such as KASP technique or PCR amplification.

[0074] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and substance thereof are within the scope of this application. Unless otherwise specified, the examples are conducted under conventional experimental conditions, such as those described in Sambrook et al.'s *Molecular Cloning: A Laboratory Manual* (Sambrook J & Russell DW, 2001), or according to the conditions recommended in the manufacturer's instructions. Unless otherwise specified, the chemical reagents used in the examples are all commercially available and conventional methods well known to those skilled in the art.

[0075] Example 1: Identification of moisture content (QTL qKDR1) in maize kernels

[0076] Using a population of 201 recombinant inbred lines derived from the cross between maize inbred lines DAN340 and K22, QTL loci for maize kernel dehydration rate were detected. The DAN340 / K22 recombinant inbred line population was planted in five geographical locations in China using a randomized block design: 2013, Hainan (Sanya; 109.19°E, 18.38°N); 2014, Hubei (Wuhan; 114.32°E, 30.58°N), Henan (Xinxiang; 113.81°E, 35.20°N), Liaoning (Shenyang; 123.47°E, 41.68°N), and Jilin (Gongzhuling; 124.83°E, 43.51°N). Kernel moisture content was measured at five consecutive stages at 34, 40, 46, 52, and 58 days post-pollination, and the area under the moisture content curve (AUDDC) was calculated to measure the kernel dehydration rate. A mixed linear model was used to calculate the best linear unbiased predictor value (BLUP) to eliminate the influence of environmental factors. The BLUP value for each strain was used as the phenotypic value for QTL mapping. Ultimately, a major QTL was detected on the left arm of chromosome 1, namely... qKDR1 This QTL explains 9.81% of the phenotypic variation. Fine mapping. qKDR1 In this phase, heterozygous individuals are planted to screen for new recombination events, using qKDR1 Flanking markers of the region were used to identify new recombinants, and novel molecular markers were developed to determine the breakpoints of identified recombinants. For new recombinants, the Student's Test was used to assess the NILs of the progeny. DAN340 and NIL K22 The dehydration rates of homozygous individuals were compared. By integrating QTL localization information from all recombinants, the QTL was narrowed down to a 1417 bp non-coding region ( Figure 1 This segment corresponds to Chromosome 1:20,007,756-20,009,147 of the B73 V4 genome. (For use in...) qKDR1 The markers and sequences for fine-tuning are shown in Table 1. Because this region is located in an intergenic region, it is neither transcribed nor translated. Therefore, this region can affect the expression levels of other genes, thus affecting the grain moisture content.

[0077] Example 2: ZmRPG gene identification

[0078] The inventors further utilized two NIL materials (named R1-R4, genotype information see) that showed separation and two that did not separate in the target region. Figure 3 A) Grains from late-mature stages were selected for RNA-seq analysis to identify differentially expressed genes. Through combined analysis of multiple RNA-seq data sets, a total of 17 differentially expressed genes were identified. Figure 2 R1 and R3 are in qKDR1 If the segment does not separate, the differentially expressed genes it produces should not be the target genes; R2 and R4 in qKDR1 Segment separation, the differentially expressed genes it produces should include qKDR1 Regulated genes. Therefore, differentially expressed genes can be identified in this way. ZmRPG ,lie in qKDR1 Approximately 10 kb downstream ( Figure 1 Further qPCR was used to verify the expression levels of ZmRPG in populations R1–R4, and it was found that in populations R2 and R4, ZmRPG In NIL DAN340 The expression level in genotyped materials is higher than that in NIL. K22 Expression levels in genotyped materials. This indicates... qKDR1 Fragments can affect ZmRPG Gene expression ( Figure 3 Due to NIL K22 Compared to NIL DAN340 It has a faster grain dehydration rate, therefore, ZmRPG The higher the expression level, the slower the grain dehydration rate; the lower the expression level, the faster the grain dehydration rate. Comparison of near-isogenic sequences revealed NIL. DAN340 In ZmRPG The full-length gene is 2010 bp (SEQ ID NO. 1), NIL K22 In ZmRPG The full-length gene is 2013 bp (SEQ ID NO. 2). These two sequences have only a few base variations, but these base variations do not affect the function of the gene.

[0079] ZmRPG The gene is located in Chromosome 1: 20,019,106-20,021,118 in the B73 V4 genome, with a total length of 2013 bp. ZmRPG The gene was not annotated on the B73-V4.0 reference genome, but transcriptional signals were present. The inventors further performed sRNA-seq and Ribo-seq on late-mature seeds, finding that this region was not covered by small RNA. Combined with ORF annotation of the unknown sequence, it was found that ZmRPG could be annotated into 12 ORFs, all of which are small peptides of only a few dozen amino acids. Figure 4Furthermore, Ribo-seq reads were found to cover the positions of ORF1, ORF2, and ORF3, indicating that they can be translated into proteins. Further analysis within the association population confirmed that only ORF1 (named microRPG1) is a small peptide protein that affects the dehydration rate of maize kernels. The nucleotide sequence encoding microRPG1 is 96 bp long (SEQ ID NO. 3), and the amino acid sequence is 31 amino acids long (SEQ ID NO. 4).

[0080] Example 3: Manipulating ZmRPG to change the dehydration rate of corn kernels

[0081] Since different expression levels of ZmRPG regulate different dehydration rates in maize kernels, the inventors further explored the use of ZmRPG in the maize inbred line B104. ubiquitin ZmRPG (SEQ ID NO. 1 and SEQ ID NO. 2) was overexpressed via promoter to verify its efficacy, using the commonly used nos terminator. Two overexpression lines, RPG-OE1 and RPG-OE2, were generated. Compared to the wild-type control, both RPG-OE1 and RPG-OE2 exhibited slower dehydration rates. Figure 5 This indicates that increasing ZmRPG expression can reduce the dehydration rate of maize kernels.

[0082] Using a similar method, microRPG1 was overexpressed in the maize inbred line B104 (using the same promoter and terminator as above), resulting in two overexpression lines, ORF1-OE1 and ORF1-OE2. Compared to the wild-type control, ORF1-OE1 and ORF1-OE2 also exhibited slower dehydration rates. Figure 6 This indicates that increasing microRPG1 can also reduce the dehydration rate of corn kernels.

[0083] This invention further utilizes CRISPR-Cas9 technology to knock out microRPG1 in maize inbred line B104. The designed target sequence is shown in SEQ ID NO. 5, and the gRNA sequence that recognizes the target is shown in SEQ ID NO. 6. For the remaining maize gene editing procedures, please refer to Example 2 of CN112646013A.

[0084] Gene editing resulted in three mutant types: KO-1bp (SEQ ID NO. 7), KO-2bp (SEQ ID NO. 8), and KO-4bp (SEQ ID NO. 9). Compared to the wild type, KO-1bp, KO-2bp, and KO-4bp knockouts all exhibited faster dehydration rates. Figure 7This indicates that knocking out microRPG1 can increase the dehydration rate of maize kernels. Example 4: Role of microRPG1 peptides in other plant species.

[0085] To further investigate the function of microRPG1, microRPG1 (named ORF1p) was chemically synthesized based on the amino acid sequence (SEQ ID NO. 4) of the micropeptide. A 2 μM aqueous solution was prepared and used to treat the model plant Arabidopsis thaliana by in vitro spraying or direct addition to the soil. Treatment was performed at the onset of silique maturation and at 50% silique maturity. A random sequence with the same amino acid composition as the microRPG1 peptide (named scORF1p) was used as a control peptide. The results showed that ORF1p delayed silique maturation (but did not change the flowering period) regardless of whether treatment was performed at the onset of silique maturation or at 50% silique maturity, and significantly increased seed water content. However, treatment with scORF1p and water did not change silique maturation or water content. Figure 8 ).

[0086] This invention also included a concentration gradient experiment (from 0.01 μM to 2 μM) to test the effect of ORF1p on the maturation of Arabidopsis thaliana siliques, with the treatment method described in the previous paragraph. The results showed that 1 μM and 2 μM of ORF1p peptides could delay the maturation of Arabidopsis thaliana siliques. Figure 9 ).

[0087] Furthermore, this invention synthesized a fluorescently labeled microRPG1 peptide (named FAM-ORF1p). FAM-ORF1p was applied to Arabidopsis thaliana in vitro at a concentration of 2 μM. Fluorescence microscopy revealed that FAM-ORF1p could be absorbed by the roots of Arabidopsis thaliana and transported to the cotyledons of the aboveground parts. Figure 10 This indicates that microRPG1 peptides can be absorbed by plant roots to influence plant growth and development, and can enter plant cells.

[0088] The above results indicate that microRPG1 peptides can influence plant growth, development, and maturation across species. Therefore, manipulating microRPG1 or applying microRPG1 peptides in vitro can alter growth and development processes, especially delaying maturation. Example 5: Anti-aging effects of microRPG1 peptides in other species.

[0089] This invention further tests the anti-aging effects of microRPG1 peptides on other non-plant species.

[0090] The chemically synthesized peptide ORF1p was co-cultured with 293T cells (human embryonic kidney cell-derived cell line) at concentrations of 20 μM and 50 μM in cell culture medium. Cell viability was measured using a CCK-8 assay kit (BCCK1000), and it was found that ORF1p significantly improved 293T cell viability. Next, 293T cells were treated with 200 μM H2O2 (hydrogen peroxide) and 50 μM ORF1p. Specifically, 293T cells were stimulated with 200 μM H2O2 for 4 h and then cultured for 24 h to construct a cellular oxidative stress model. In the experimental group, ORF1p was added to the culture medium (final concentration 50 μM), while the control group received either an equal volume of double-distilled water or no ORF1p. Both groups were co-cultured for 24 h. The reactive oxygen species (ROS) content was then measured using a reactive oxygen species (ROS) detection kit. It was found that compared with the control group, ORF1p has the potential function of reducing ROS production at the cellular level and alleviating oxidative stress. Figure 11 This result indicates that microRPG1 peptides also have anti-aging functions in human cells.

[0091] The human homolog of *C. elegans* is approximately 60%-80%, containing at least 42% of human disease-related genes (Sugiet et al., 2016). These advantages make... C. elegans As a widely used animal model, it is often used to study directions related to anti-aging.

[0092] Further treatment of the model animal with 50 μM ORF1p at a concentration of 50 μM was performed on the hermaphroditic N2 wild-type Caenorhabditis elegans (C. elegans). Caenorhabditis elegans , C . elegans The study set up a treatment group and a control group, with 7 replicates in each group. Each replicate treated 20 worms. The treatment method involved thoroughly mixing E. coli OP50 bacterial suspension and the small peptide, then spreading the mixture onto a petri dish. The bacterial culture was repeated weekly by shaking to ensure the effectiveness of the small peptide. The worms were incubated at 20°C for at least 40 days. Direct observation revealed that the aggregation of intestinal particles in nematodes fed ORF1 appeared later than in the control group. Figure 12 Intestinal particulate matter aggregation is an important indicator associated with nematode aging. Studies on nematode lifespan showed that the ORF1p treatment group had an average lifespan approximately 13.2% longer than the control group. Figure 13 ).

[0093] Further investigation involved intravenously injecting mice with 100 μL of 0.9% saline containing 0.2 μg–20 μg of ORF1p, and observing the mice's performance after injection of the micropeptide. The results showed that ORF1p also increased the lifespan of the mice. In addition, feeding fruit flies with micropeptide-added culture media at three different concentrations (2 µM, 10 µM, and 50 µM) revealed that the micropeptide also prolonged the lifespan of the fruit flies. The culture medium used to feed the fruit flies contained the following components: 66.825 g corn flour, 9.18 g soybean flour, 6 g agar, 40 g sucrose, 42.4 g maltose, 25 g yeast, 6.85 mL propionic acid, 1 g sodium benzoate, 0.25 g methylparaben, and different amounts of micropeptide, finally bringing the volume to 1 L.

[0094] Example 6: Identification of microRPG1 homologous proteins in Tetrapanax papyrifer.

[0095] The inventors searched for homologous sequences of the microRPG1 peptide in Zea luxurians by sequence alignment, and identified eight similar peptides (sequences shown as SEQ ID NO. 11~18) in eight different wild Zea luxurians species (Zea luxurians-RIL003, Zea nicaraguensis-PI615697, Zeadiploperennis-Gigi, Zea diploperennis-Momo, Zea perennis, Zea mays subspecieshuehuetenangensis-RIMHU001, Zea mays subspecies parviglumis, Zea mayssubspecies mexicana-TIL25).

[0096] The inventors further synthesized homologous micropeptides found in bulrush and tested their functions according to the methods in Examples 4 and 5. They found that these eight micropeptides, like microRPG1 in corn, can also affect the grain water content of plants and have the effect of delaying the aging of plants and animals.

[0097] Therefore, these microRPG1 peptides can be developed into cosmetic raw materials, pharmaceuticals, health products, plant nutrient solutions, and other products for use in areas such as increasing the water content of plant seeds and anti-aging.

[0098] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A micropeptide, characterized in that, The amino acid sequence of the micropeptide is shown in SEQ ID NO.

4.

2. A formulation, characterized in that, The micropeptide of claim 1 is dissolved in a specific reagent at a concentration of 1 μM to 50 μM; The reagents include: water and physiological saline.

3. The use of the peptide with the amino acid sequence shown in SEQ ID NO. 4, characterized in that, The use includes any of the following: 1) Increase the moisture content of corn or Arabidopsis thaliana kernels; 2) Delay the ripening of corn or Arabidopsis thaliana fruits; 3) Prepare formulations to delay aging, prolong lifespan, or reduce reactive oxygen species generation.

4. The application of a nucleic acid molecule in improving the moisture content or dehydration rate of corn kernels, characterized in that, The nucleotide sequence or reverse complementary sequence of the nucleic acid is shown in any one of SEQ ID NO. 1 to SEQ ID NO.

3.

5. A method for reducing the moisture content of corn kernels or increasing the dehydration rate, characterized in that: Inhibit the expression and / or activity of the micropeptide of the sequence shown in SEQ ID NO. 4 or any one of the sequences shown in SEQ ID NO. 1 to SEQ ID NO. 3 in maize, and select maize plants with reduced kernel moisture content or increased dehydration rate.

6. The method according to claim 5, characterized in that, The methods for inhibiting the expression and / or activity of micropeptides or nucleic acid molecules include gene editing or RNA interference.

7. The method according to claim 6, characterized in that, The target DNA sequence for gene editing is shown in SEQ ID NO.

5.

8. A kit for reducing the moisture content of corn kernels or increasing the dehydration rate, characterized in that, Including any of the following: (1) An RNA molecule capable of recognizing the target sequence described in claim 7; (2) The DNA molecule encoding the RNA described in (1); (3) A vector for expressing the RNA described in (1).

9. The reagent kit according to claim 8, characterized in that, The sequence of the RNA molecule is shown in SEQ ID NO.

6.

10. A mutant gene, characterized in that: The nucleic acid sequence of the mutated gene is shown in any one of SEQ ID NO. 7 to SEQ ID NO.

9.

11. A method for increasing the moisture content of corn kernels or reducing the dehydration rate, characterized in that, Increase the expression and / or activity of the micropeptide of the sequence shown in SEQ ID NO. 4 or any one of the sequences shown in SEQ ID NO. 1 to SEQ ID NO. 3 in the maize material to be improved, and select plants with increased maize kernel moisture content or decreased dehydration rate.

12. The method according to claim 11, characterized in that, The method for enhancing expression and / or activity is to drive nucleic acid molecule expression using a highly active promoter.

13. The method according to claim 12, characterized in that, The highly active promoter is the corn ubiquitin promoter.

14. The method according to claim 13, characterized in that, The corn ubiquitin promoter sequence is shown in SEQ ID NO.

10.

15. The method of any one of claims 5-7, or the kit of any one of claims 8-9, or the mutant gene of claim 10, or the method of any one of claims 11-14, in improving the moisture content or dehydration rate trait of maize kernels.

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