CN114685635A - Gene FLO18 for regulating development and quality of endosperm - Google Patents

Abstract

The present invention provides a gene FLO18 that regulates the development and quality of endosperm. The inventors cloned and obtained a novel gene FLO18 related to the regulation of plant yield traits or endosperm quality, which is a positive regulator of yield or endosperm quality. decreased, but had no effect on other plant traits. The present invention provides a new target for the improvement of plant traits.

Description

Gene FLO18 regulating endosperm development and quality

technical field

The present invention belongs to the fields of biotechnology and botany, and more particularly, the present invention relates to the gene FLO18 that regulates the development and quality of endosperm.

Background technique

The endosperm of angiosperms develops after double fertilization with one sperm cell and two polar nuclei located in the central cell, and is a triploid tissue. The developmental process of plant endosperm can be divided into three types: nuclear endosperm, cellular endosperm, and swamp endosperm.

Poaceae rice, as one of the most important food crops in the world, has a huge market and demand. Rice consumed by people is the endosperm part of rice, which accounts for 70-80% of the grain. Meanwhile, rice endosperm plays an important role in rice embryo formation and seed germination. Proving the genetic development mechanism of endosperm and revealing the relationship between rice endosperm development and rice yield and quality is of great significance for improving rice yield and quality.

The endosperm development process of rice belongs to the nuclear endosperm type. That is, in the early stage of endosperm development, only nuclear division is carried out without cytoplasmic division, and no cell wall is formed. Cytoplasmic division and cell wall formation occur only when development reaches a certain level.

The development of rice endosperm involves mitosis, cellularization, cell differentiation, accumulation of storage substances, and programmed death, and the entire development is generally completed within 21 to 22 days after fertilization. The period of 0-2 DAP (Days After Pollination) is mainly for nuclear division but no cytokinesis, 3-5 DAP is mainly for cellularization, and 6-21 DAP is mainly for endosperm cell differentiation and accumulation of storage substances. At 8DAP, programmed cell death gradually spread from the center of the endosperm to the entire endosperm, and at 21DAP, only the aleurone cells remained active. From 22 to 30 DAP, the fresh weight and dry weight of rice caryopsis no longer increased. In the starch endosperm, complex starch granules were formed along with the blurring of the edges of individual starch granules. At the whole grain level, crystallization of starch and storage results in the formation of a translucent starchy endosperm at the end of the maturation process.

After the rice endosperm is fully mature, the remaining dry matter after dehydration is 80-90% starch, 7-9% protein, 0.3-0.5% lipid, and also contains potassium, magnesium, calcium, zinc and other inorganic substances.

There are two types of starch - amylose and amylopectin. Amylose is linked by α(1-4) glycosidic bonds, and there are about 200 glucoses in each molecule; in addition to α(1-4) glycosidic bonds, amylopectin is also linked by α(1-6) glycosidic bonds. Branched structure; branched chains and amylose are often stained with iodine solution, amylose is blue in the presence of iodine, and amylopectin is red. The starch in the endosperm of general varieties of rice consists of 20-30% amylose and 70-80% amylopectin.

In rice, starch is mainly synthesized by four types of enzymes: adenosine diphosphate glucose pyrophosphorylase (ADP-glucose pyrophosphorylase, AGPase), starch synthase (Starch synthases, SS), granule bound starch synthase (granule bound starch synthase) SS, GBSS), Starch branching enzymes (SBE), Starch debranching enzymes (DBE).

In the endosperm, AGPase in the cytoplasmic matrix catalyzes the formation of ADP-glucose from glucose-1-phosphate and ATP, releasing PPi; ADP-glucose is the raw material for starch synthesis, which is the first key step in starch synthesis. Starch synthase SS catalyzes the transfer of the glucosyl group of ADP-glucose to the non-reducing end of the preexisting α(1-4) glycosidic linkage-linked glucan primer to form amylose and amylopectin. The main function of starch branching enzyme SBE is to cleave α-(1-4) glycosidic bond, transfer the released reducing end to C6 hydroxyl end to generate α-(1-6) glycosidic bond, and form branched structure of amylopectin molecule. Starch debranching enzyme DBE is mainly used to hydrolyze abnormally branched α-(1-6) glycosidic bonds in amylopectin, to adjust the branching of amylopectin and maintain the crystallinity of amylopectin. In addition, disproportionating enzyme (DPE) and starch phosphorylase (phosphorylase, PHO) play important roles in the initiation and elongation of starch multimers.

These types of enzymes synergize to synthesize starch in rice, hydrolyze abnormal starch structure, form starch granules in starch body, and finally form starch crystal structure with compact polyhedron structure, any one of these enzymes changes, or Changes in the related genes that regulate these enzymes will affect the synthesis and accumulation of starch and affect the quality of rice grains.

For example, changes in the gene encoding the AGPase subunit lead to abnormal starch granules in the rice endosperm and a decrease in starch content. Alterations in the GBSSI gene affect starch synthesis, producing waxy endosperm. Mutation of OsSSIIIa results in a white-hearted endosperm, which affects the physicochemical properties of starch in the endosperm. In addition, mutations in different subfamilies of starch synthase and starch branching enzymes, such as OsSSI, OsBEI, and OsBEIIb, all resulted in the silty endosperm trait.

In addition to enzymes involved in starch synthesis, changes in genes involved in other metabolic pathways also indirectly affect starch synthesis, resulting in abnormal endosperm. Both OsPK2 and OsPKα1 encode a plastid pyruvate kinase, and OsPPDK encodes a pyruvate phospho-dikinase, both of which belong to the glycolytic pathway, but affect the formation of starch granules and grain filling. Du1 encodes a member of the pre-mRNA processing protein family, which is mainly expressed in rice panicle and regulates starch synthesis by specifically affecting the silencing of Wxb gene. Wx gene is a major QTL locus controlling amylose content in rice. PFP1 encodes a beta subunit of fructose pyrophosphate 6-phosphate 1-phosphotransferase, which converts fructose 6-phosphate and pyrophosphate to fructose 1,6 in the glycolytic pathway Diphosphate, PFP1, in the form of a heterotetramer, reversibly regulates the metabolic flux of endosperm, and regulates starch synthesis and grain development. FLO15 encodes a plastid glyoxalase (OsGLYI), which is mainly involved in the detoxification process of methylglyoxal. Changes in OsGLYI can lead to changes in the expression of genes related to starch synthesis, resulting in the production of fenugreek endosperm. FLO11 encodes a plastid heat shock protein (OsHsp70cp-2) whose mutation results in white heart and peripheral farin of rice grains; this gene is abundantly expressed in the developing endosperm, and OsHsp70cp-2 interacts with the Tic complex to regulate the protein Transport of amyloplasts. FLO16 encodes an NAD-dependent cytosolic malate dehydrogenase whose primary function is to control redox homeostasis in rice endosperm, which is important for both starch granule formation and subsequent starch synthesis. Thousand kernel weight was significantly increased in rice overexpressing FLO16.

Some genes that indirectly affect starch synthesis encode proteins with specific structures. The nuclear-localized gene FLO2 has a TPR binding site structure and positively regulates the expression of starch synthesis genes by binding to a transcription factor with a basic helix-loop-helix (bHLH) structure. FLO6 is a protein with a carbohydrate-binding domain (CBM) that regulates starch synthesis and starch granule formation by interacting with starch isomerase (ISA1). OsNPPR1 is a nuclear-localized pentatricopeptie repeat protein (PPR), which encodes a protein that binds to CUCACmotif, regulates the splicing of specific introns, and affects mitochondrial development in endosperm, which in turn affects endosperm development. FLO10 also encodes a triangular-shaped pentapeptide repeat protein (pentatricopeptierepeat protein, PPR), which is encoded by nuclear genes and localized to mitochondria, and is mainly involved in the trans-silencing of the first intron of mitochondrial nad1, because nad1 encodes NADH in mitochondria Changes in FLO10, a core component of complex I of the electron transport chain, affect the correct splicing of nad1 and affect endosperm development to form a silty endosperm. In addition, OsNDUFA9, OsAlAT1, OsBT1 and other genes indirectly regulate starch synthesis in endosperm by regulating starch synthase.

The protein in rice endosperm is mainly composed of gliadin, globulin, glutenin and albumin, among which glutenin accounts for about 80% of the storage protein. Protein body I (PBI) and protein body II (PBII) are mainly stored in the endosperm. After protein synthesis, gluten, globulin and albumin are stored in vacuoles through the vacuolar sorting pathway, and finally develop into PBII, which has no fixed shape; gliadin is stored in the endoplasmic reticulum through the endoplasmic reticulum residency pathway, and finally develops Into PBI, PBI is spherical vesicle.

There has been some progress in the regulation of gluten trafficking in existing studies, and gluten precursors are synthesized in the rough endoplasmic reticulum via a dense vesicle-mediated post-Golgi transport pathway or an endoplasmic reticulum-derived precursor accumulation lumen. Transfer to PBII. The abnormal accumulation of gluten precursors can lead to the appearance of powdery endosperm in rice endosperm and affect rice quality. For example: GOT1B encodes a Golgi transporter protein that, as a component of COPII, regulates the transport of proteins from the endoplasmic reticulum to the Golgi apparatus. The protein encoded by GPA3 acts like a scaffold, recruiting the guanine nucleotide exchange factor OsVPS9A. VPS9A in turn activates the small G protein OsRab5a. GPA3, OsVPS9A, and OsRab5a combine to form a complex on dense vesicles to regulate vacuolar trafficking of gluten precursors. PDIL1-1 encodes a disulfide isomerase-like enzyme that localizes to the rough endoplasmic reticulum and regulates the formation of disulfide bonds within the endoplasmic reticulum. Gup3 encodes a vacuolar processing protein that primarily functions to process gluten precursors into acidic and basic subunits in PBII. OsNHX5 encodes a Na+/H+ antiporter, localized in the Golgi apparatus, trans-reticular, and maintains pH homeostasis mainly in the prevacuolar lumen, and plays a very important role in gluten transport mediated by dense vesicles.

In mature rice endosperm, lipids only account for a very small part of the endosperm. However, lipid changes can affect the appearance and quality of endosperm. For example: FSE1 (floury shrunken endosperm1) encodes a phospholipase-like protein, which is localized in the cytoplasmic matrix and intracellular membrane. When FSE1 is mutated, starch granules in rice endosperm develop abnormally, the content of total starch and amylopectin decreases, lipid and The total protein content increased. Studies have shown that FSE1 mainly controls the synthesis of galactolipids in rice endosperm, and is also the link between lipid metabolism and starch synthesis. OsLTPL36 encodes a lipid transporter that is specifically expressed in the developing seed coat and aleurone. Down-regulation of OsLTPL36 expression resulted in decreased rice seed setting rate and 1000-grain weight. Inhibition of OsLTPL36 expression not only leads to a decrease in fat content, but also leads to delayed development of rice embryos.

The cytoskeleton plays an important role in the cell division and differentiation of the endosperm and the accumulation of substances. Changes in the structure and function of microscopic and related microscopic binding proteins in the cytoskeleton will affect the grain shape and endosperm quality of rice. For example, the rice MT localization protein OsIQ67-DOMAIN14 (OsIQD14) is highly expressed in rice seed husk cells. When OsIQD14 is deficient, seeds are short and wide, resulting in increased overall yield, whereas overexpression results in narrow and long seeds. OsIQD14 regulates mediated MT reordering by affecting MT dynamics, thereby regulating local cell morphology in rice grains and altering rice yield traits.

In maize, Vks1 encodes ZmKIN11, which belongs to the kinesin-14 subfamily and is mainly expressed in the early endosperm development. VKS1 is dynamically localized to the nucleus and microtubules and plays a key role in the migration of cells to free nuclei during early mitosis and cytokinesis. Absence of VKS1 is not lethal, but causes malformations such as spindle assembly, sister chromatid segregation, and septum formation resulting in decreased cell proliferation and thus different seed sizes.

Although there has been much research and attention on the endosperm development of plants in the art, since rice and other plants are important food crops, it is urgent in the art to find many genes that affect the endosperm development of plants, so as to facilitate further plant breeding and improvement.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a novel gene FLO18 that controls the development and quality of endosperm.

In the first aspect of the present invention, there is provided the use of a FLO18 protein or a regulatory molecule thereof for regulating the yield traits, endosperm traits or starch development traits of Poaceae.

In a preferred embodiment, the FLO18 protein includes its homologue.

In another preferred embodiment, the regulatory molecule is an up-regulated molecule, and the use of the FLO18 protein or its up-regulated molecule includes selected from: (i) increasing yield; preferably including increasing the grain width or grain thickness of grains, or promoting Grain filling; (ii) improving endosperm quality; preferably including promoting equal division of cells during endosperm development, or promoting the formation of horny endosperm; (iii) promoting starch development; preferably including increasing starch granule diameter.

In another preferred embodiment, the up-regulated molecule comprises: an expression cassette or an expression construct (including an expression vector) that overexpresses the FLO18 protein; or, an expression cassette or an expression construct that improves the translation efficiency of the FLO18 protein; Or, an up-regulated molecule that interacts with the FLO18 protein to increase its expression or activity.

In another preferred embodiment, the regulatory molecule is a down-regulating molecule, and its uses include: (i) reducing yield; preferably reducing grain width or grain thickness, or reducing grain filling; (ii) reducing Endosperm quality; preferably includes inhibiting the equal division of cells during endosperm development, or promotes the formation of powdery endosperm; (iii) interferes with starch development; preferably includes reducing the diameter of starch granules.

In another preferred embodiment, the down-regulated molecule includes: an agent for knocking out or silencing the gene encoding FLO18 protein, and an agent for inhibiting the activity of FLO18 protein; preferably, the down-regulated molecule includes: a gene encoding the FLO18 protein A gene editing reagent, a homologous recombination reagent or a site-directed mutagenesis reagent, the reagent performs loss-of-function mutation on the FLO18 protein; or, an interfering molecule that specifically interferes with the expression of the gene encoding the FLO18 protein.

In another aspect of the present invention, there is provided a method for regulating yield traits, endosperm traits or starch development traits of grasses, comprising: regulating the expression or activity of FLO18 protein in plants.

In a preferred example, the regulation is to increase yield, improve endosperm quality or promote starch development; the method includes: up-regulating the expression or activity of FLO18 protein in plants.

In another preferred embodiment, the regulation is to reduce yield, reduce endosperm quality or interfere with starch development; the method comprises: down-regulating the expression or activity of FLO18 protein in plants.

In another preferred embodiment, the up-regulating expression or activity of FLO18 protein in plants comprises: overexpressing FLO18 protein in plants; or, regulating with an up-regulated molecule that interacts with FLO18 protein, thereby increasing the expression of FLO18 protein or active.

In another preferred embodiment, the down-regulation of the expression or activity of FLO18 protein in plants includes: knocking out or silencing the gene encoding FLO18 protein in plants, or inhibiting the activity of FLO18 protein; preferably, it includes: using a CRISPR system to perform Gene editing to knock out the gene encoding the FLO18 protein; knock out the gene encoding the FLO18 protein by homologous recombination; silence with an interfering molecule that specifically interferes with the expression of the gene encoding the FLO18 protein; or mutate the FLO18 protein with loss of function.

In another preferred embodiment, the gramineous plant is a plant expressing FLO18 protein or its homologue; preferably, the gramineous plant is selected from (but not limited to): rice, barley, wheat, oat, Rye, corn, sorghum, Brachypodium.

In another preferred embodiment, the grasses are plants with relatively low expression of FLO18 protein (such as significantly lower than the expression level of the gene in this plant) or no expression, which can improve the expression or activity of FLO18 protein by increasing the expression or activity of FLO18 protein. Increase yield, increase grain width or grain thickness, promote grain filling, promote starch development, increase starch granule diameter, promote the formation of horny endosperm, or promote equal cell division during endosperm development.

In another preferred embodiment, the amino acid sequence of the polypeptide of FLO18 is selected from the following group: (i) a polypeptide having the amino acid sequence shown in SEQ ID NO:2; (ii) the amino acid sequence shown in SEQ ID NO:2 The sequence is formed by the substitution, deletion or addition of one or several (such as 1-20, 1-10, 1-5, 1-3) amino acid residues, and has (i) polypeptide function and is composed of (i) a derived polypeptide; (iii) the homology of the amino acid sequence to the amino acid sequence shown in SEQ ID NO: 2 is ≥85% (preferably ≥90%, ≥95%, ≥98% or ≥99%), A polypeptide having the function of regulating properties; (iv) an active fragment of the polypeptide having the amino acid sequence shown in SEQ ID NO: 2; or (v) adding a tag sequence to the N or C terminus of the polypeptide having the amino acid sequence shown in SEQ ID NO: 2 Or an enzyme cleavage site sequence, or a polypeptide formed by adding a signal peptide sequence to its N-terminus.

In another aspect of the present invention, the use of FLO18 protein or its encoding gene is provided as a molecular marker for identifying traits of grasses; the traits include: yield traits, endosperm traits or starch development traits.

In another aspect of the present invention, there is provided a method for directional selection or identification of grasses with high yield, high endosperm quality or good starch development, comprising: identifying the expression or activity of FLO18 protein in the test plant, if the test plant is The expression or activity of FLO18 protein is higher than or equal to the average value of FLO18 protein expression or activity in this type of plant (control plant), then it is high yield, high endosperm quality or good starch development (such as full grain, good grain filling, starchy Well-developed grasses with large diameter of starch granules or capable of forming horny endosperm).

In a preferred example, the high expression or high activity refers to a statistically significant increase in the expression or activity compared with the average value of the expression or activity of the same or the same plant, such as an increase of 10%, 20%, 40%, 60%, 80%, 90% or higher.

In another preferred embodiment, the low expression or low activity refers to a statistically significant decrease in expression or activity compared with the average value of the expression or activity of the same or the same plant, such as a decrease of 10%, 20% , 40%, 60%, 80%, 90% or less.

In another preferred example, the "high amount"/"high quality (or good)" refers to a statistically significant higher amount compared with the amount of the same or the same plant, such as 10%, 20%, 40%, 60%, 80%, 90% or higher.

In another preferred embodiment, the low amount refers to a statistically significant lower amount compared with the amount of the same or the same plant, such as 10%, 20%, 40%, 60%, 80%, 90% lower or lower.

In another aspect of the present invention, there is provided a method for screening substances (potential substances) that promote the improvement of grass plant traits, the trait improvement comprising: high yield, high endosperm quality or good starch development; the method includes: ( 1) Add the candidate substance to the system expressing the FLO18 protein; (2) Detect the system, and observe the expression or activity of the FLO18 protein. %, 60%, 80%, 90% or higher), it indicates that the candidate substance is a substance that promotes the improvement of grass plant traits.

In a preferred example, the method further includes: setting a control group without adding the candidate substance, so as to clearly distinguish the difference between the expression or activity of the FLO18 protein in the test group and the control group.

In another preferred example, the candidate substances include (but are not limited to): regulatory molecules (such as up-regulators, small-molecule compounds, gene editing constructs) designed for the FLO18 protein or its encoding gene or its upstream or downstream proteins or genes. things etc.

In another aspect of the present invention, there is provided a Gramineae cell, tissue or organ, which contains an exogenous FLO18 protein up-regulated molecule, wherein the up-regulated molecule includes an expression cassette or an expression cassette that overexpresses the FLO18 protein. constructs (including expression vectors); or expression cassettes or expression constructs that increase the translation efficiency of the FLO18 protein; or up-regulated molecules that interact with the FLO18 protein to increase its expression or activity.

In a preferred example, the plant cells, tissues or organs do not have the ability to reproduce.

Other aspects of the invention will be apparent to those skilled in the art from the disclosure herein.

Description of drawings

Figure 1a, a schematic diagram of the process of hybridizing mutant material XA80 with 9311 to construct a targeting population.

Figure 1b. Determination of mutation sites in mutants compared to wild type.

Fig. 1c, Phylogenetic tree constructed by Neightbor-joining method.

Figure 2a. Phenotypic investigation of mutant and wild-type plants, the left side is the wild type, and the right side is the mutant bar=20cm.

Figure 2b. The grains of the mutant and wild-type plants were obtained, and the appearance was compared after the glumes were removed (left picture) and observed under a stereomicroscope (right picture). bar=1 cm.

Figure 2c. Thousand kernel weight (left panel) and grain filling rate (right panel) of mutant and wild-type plants.

Figure 2d. Comparison of grain width (upper panel) and kernel thickness (lower panel) of mutant and wild-type plants.

Fig. 2e, SEM images of whole grain, cross-section and starch granule state of mutant (lower panel) and wild-type (upper panel) plants.

Figures 2f~g, photos of cellular structure of endosperm under optical microscope (f) and transmission electron microscope (g).

Figure 3a. Nuclei in the perivascular cells of mutant and wild-type plants in endosperm 6 days after pollination.

Figure 3b. The expression position of FLO18 in cells was observed under Zeiss LSM880 laser confocal microscope.

Figure 3c, Co-transfection experiment of FLO18 and TUB6.

Detailed ways

After extensive research, the inventors cloned and obtained a novel gene FLO18, which is related to the regulation of plant yield traits or endosperm quality. Phenotype, yield and quality decreased, but no effect on other plant traits. The present invention provides a new target for the improvement of plant traits.

the term

As used herein, "plant" includes plants expressing or comprising FLO18 and the signaling pathways in which it is involved. According to the knowledge in the art, plants expressing FLO18, which have the mechanism of action as claimed in the present invention, can achieve the technical effects as claimed in the present invention. In some preferred modes, the plant is a crop, preferably a cereal crop, and the cereal crop is a crop with grain (ear grain). The "grain crops" may be grasses. Preferably, the grasses include: rice, barley, wheat, oats, rye, corn, sorghum, Brachypodium and the like.

As used herein, the terms "improve", "improvement" or "enhancement" are interchangeable with each other and shall in the applied sense mean at least 2%, 3%, 4%, 5%, as compared to a control plant as defined herein %, 6%, 7%, 8%, 9% or 10%, preferably at least 15% or 20%, more preferably 25%, 30% higher adjustment.

With regard to "control plants", selection of appropriate control plants is a routine part of experimental design and can include corresponding wild-type plants or corresponding transgenic plants without the gene of interest. Control plants are generally the same plant species or even varieties of the same or the same class as the plants to be evaluated. Control plants can also be individuals that have lost the transgenic plant by segregation. Control plants as used herein refer not only to whole plants, but also to plant parts, including seeds and seed parts.

As used in the present invention, the "grain" refers to the fruit or seed of a plant, and is also called ear grain in crops such as rice, corn, wheat, and barley.

FLO18 gene and its encoded protein

In the present invention, unless otherwise specified, the FLO18 protein includes its homologue (homologous protein). The FLO18 is a polypeptide (protein) with the amino acid sequence shown in SEQ ID NO:2. The present invention also includes sequence variant forms that have the same function as the FLO18 protein.

The variant forms include (but are not limited to): several (usually 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10, still better Deletion, insertion and/or substitution of amino acids such as 1-8, 1-5), and addition or deletion of one or several (usually within 20, preferably 10) C-terminal and/or N-terminal within, more preferably within 5) amino acids. Any high homology with the FLO18 protein (for example, the homology with the polypeptide sequence shown in SEQ ID NO: 2 is 70% or higher; preferably the homology is 80% or higher; more preferably Proteins having a homology of 90% or more, such as 95%, 98% or 99% homology) and having the same function as the FLO18 protein are also included in the present invention.

Polypeptides derived from species other than rice that have higher homology to the polypeptide sequence of the sequence shown in SEQ ID NO: 2, or play the same or similar roles in the same or similar signaling pathways are also included in the present invention.

In the present invention, the "FLO18 protein" also includes their homologues. It should be understood that although FLO18 proteins obtained from a specific species are preferably studied in the present invention, those obtained from other species, especially grasses, are highly homologous to said FLO18 protein (such as having more than 70%, more particularly 80%, Other polypeptides or genes with 85%, 90%, 95%, or even more than 98% sequence identity) are also within the scope of the present invention.

The present invention also provides an isolated protein, which is formed by a fragment of the FLO18 protein or by adding other proteins or tags at both ends.

The present invention also relates to a polynucleotide sequence encoding the FLO18 protein of the present invention or a sequence variant thereof. The polynucleotide may be in DNA form or RNA form. DNA forms include cDNA, genomic DNA or synthetic DNA. DNA can be single-stranded or double-stranded. DNA can be the coding or non-coding strand. The coding region sequence encoding the mature polypeptide can be identical to the coding region sequence shown in SEQ ID NO: 1 or a degenerate variant. As used herein, a "degenerate variant" in the present invention refers to a polypeptide encoding a polypeptide having the sequence of SEQ ID NO:2, but differing from the genomic sequence shown in SEQ ID NO:3 or the encoding shown in SEQ ID NO:1 A nucleic acid sequence that differs in region sequence. The present invention also relates to variants (variants) of the above-mentioned polynucleotides, which encode polypeptides or fragments, analogs and derivatives of polypeptides having the same amino acid sequence as the present invention.

The present invention also relates to vectors comprising said polynucleotides, and host cells genetically engineered with said vectors or polypeptide-encoding nucleic acids.

In the present invention, the polynucleotide sequence encoding the polypeptide of the present invention can be inserted into a recombinant expression vector. The term "recombinant expression vector" refers to bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses or other vectors well known in the art. In short, any plasmid and vector can be used as long as it is replicable and stable in the host. An important feature of expression vectors is that they typically contain an origin of replication, a promoter, marker genes and translational control elements. Preferably, resistance elements, screening (selection) elements or reporter gene elements, such as Bar and GUS, can also be selectively added to the expression vector.

When the polynucleotide is expressed in higher eukaryotic cells, transcription will be enhanced if an enhancer sequence is inserted into the vector. Enhancers are cis-acting elements of DNA, usually about 10 to 300 base pairs in length, that act on a promoter to enhance transcription of a gene.

Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. Agrobacterium transformation or biolistic transformation and other methods can be used to transform plants, such as spraying method, leaf disk method, rice immature embryo transformation method, and the like.

plant modification

Through a large number of systematic studies, the inventors cloned the full-length sequence of FLO18 and identified its biological function. This gene plays an important role in the development of rice endosperm. The development of the structure further affects the accumulation of storage substances and thus the quality of the endosperm. The yield of the FLO18 mutant was reduced but had no significant effect on the number of grains per panicle, panicle length and panicle shape. It is conducive to a deeper understanding and analysis of the role of the cytoskeleton in the development of endosperm.

In a specific example, the inventors found that the 1000-grain weight of the mutant FLO18 was significantly lower than the wild-type 1000-grain weight, the grain width was significantly smaller than the wild-type grain width, and the grain thickness was significantly smaller than the wild-type grain thickness through gene mapping population construction and phenotypic investigation. . Compared with the normal individuals, the grain filling rate of the mutant was not obvious in the first 3 days, and the dry matter accumulation gradually slowed down from the 6th day; the dry weight after the final maturity was significantly lower than that of the wild type. The mutant starch was not fully developed, the starch granules were obviously smaller, the starch granules were spherical, and the space between the starch granules was larger. In addition, the development of amyloid in the mutant was significantly slower than that in the wild type, and the crystal structure was loose and the granules were small, and the existence of protein bodies was rarely observed.

In a specific example, the inventors found that the XA80 mutant complemented by FLO18 showed the phenotype of the wild-type japonica IL-9 through the replenishment experiment, and its grain shape was plump and smooth, and its cross-section was transparent and compact; its thousand-grain weight was also Thousand-grain weight, grain width and grain thickness were basically the same as those of wild-type plants.

In a specific example, the inventors also found that in the process of endosperm development, the mutants have uneven division of nuclei, thus confirming that FLO18 is related to cell division and thus regulates endosperm quality.

Based on the new discovery of the present inventors, the use of a FLO18 protein or a regulatory molecule thereof is provided for: regulating the yield traits, endosperm traits or starch development traits of grasses.

At the same time, the present invention also provides a method for regulating the yield traits, endosperm traits or starch development traits of gramineous plants, comprising: regulating the expression or activity of FLO18 protein in plants; wherein, the FLO18 protein includes its homology thing.

It should be understood that, after knowing the role of the FLO18 protein in the regulation of the yield traits, endosperm traits or starch development traits of grasses, the various methods well known to those in the art can be used to adjust the The expression or activity of the FLO18 protein, these methods are all included in the present invention.

The activity of the FLO18 protein can be up-regulated using an up-regulator of the expression or activity of the FLO18 protein. The up-regulators of FLO18 protein expression or activity include promoters, agonists, and activators. The "up-regulation" and "promotion" include "up-regulation" and "promotion" of protein activity or "up-regulation" and "promotion" of protein expression. Any substance that can increase the activity of FLO18 protein, improve the stability of FLO18 protein gene or protein, up-regulate the expression of FLO18 protein gene, and increase the effective action time of FLO18 protein, these substances can be used in the present invention, as for up-regulating FLO18 protein or its substances. A useful substance for the encoded protein. They can be chemical compounds, small chemical molecules, biomolecules. The biomolecules can be at the nucleic acid level (including DNA, RNA) or at the protein level.

As a preferred embodiment, a method for up-regulating the expression of FLO18 protein in plants is provided, the method comprising: transforming the expression construct or vector of FLO18 protein or the protein encoded by it into plants.

Preferably, a method for preparing a transgenic plant is provided, comprising:

(1) transforming an exogenous nucleic acid encoding the polypeptide of the present invention into a plant organ or tissue to obtain a plant tissue or organ transformed into the nucleic acid encoding the polypeptide; and

(2) Regenerating the plant tissue or organ obtained in step (1) into which the exogenous nucleic acid encoding the polypeptide of the present invention has been transferred into a plant plant.

As a preferred example, the method includes the steps:

(s1) Agrobacterium carrying an expression vector is provided, and the expression vector contains the nucleic acid encoding the FLO18 protein of the present invention;

(s2) contacting the plant tissue or organ with the Agrobacterium in step (s1), so that the nucleic acid encoding the polypeptide is transferred and integrated into the chromosome of the plant cell;

(s3) selecting the plant tissue or organ into which the nucleic acid encoding the FLO18 protein is transferred; and

(s4) Regenerating the plant tissue or organ in step (s3) into a plant.

The present invention also includes plants obtained by any one of the aforementioned methods, and the plants include: transgenic plants into which the nucleic acid encoding the polypeptide has been transformed.

As an alternative way, the upstream open reading frame (upstream open reading frame; uORF) on the 5'UTR of the FLO18 gene is knocked out to improve the expression cassette or expression construct of the translation efficiency of the FLO18 gene. uORF is widely present in eukaryotic mRNA, which is a translational regulatory element. Typically, translation of uORFs takes precedence over mORFs (major open reading frames), resulting in blocked mORF translation. In the present invention, targeted knockout of the uORF on the 5'UTR of the FLO18 gene can effectively improve the translation efficiency of the FLO18 protein and improve its expression.

In the present invention, the down-regulating agent of FLO18 protein or its encoded gene refers to any agent that can reduce the activity of FLO18 protein, reduce the stability of FLO18 protein or its encoded gene, down-regulate the expression of FLO18 protein, reduce the effective action time of FLO18 protein, Substances that inhibit the transcription and translation of the FLO18 gene, or reduce the phosphorylation/activation level of the protein, can be used in the present invention as useful substances for down-regulating the FLO18 protein. They can be chemical compounds, small chemical molecules, biomolecules. The biomolecules can be at the nucleic acid level (including DNA, RNA) or at the protein level. For example, the down-regulating agents are: interfering RNA molecules or antisense nucleotides that specifically interfere with the expression of FLO18 protein or other signaling pathway genes; or gene editing reagents that specifically edit the FLO18 gene, and the like.

As a preferred mode of the present invention, there is provided a method for down-regulating FLO18 protein in plants, which comprises targeted mutation, gene editing or gene recombination of FLO18 protein to achieve down-regulation. As a more specific embodiment, by any of the above methods, the FLO18 protein is converted into its mutant, so that it no longer functions. As a more specific embodiment, the CRISPR/Cas9 system is used for gene editing. Appropriate sgRNA target sites will bring higher gene editing efficiency, so before proceeding with gene editing, suitable target sites can be designed and found. After designing specific target sites, in vitro cell activity screening is also required to obtain effective target sites for subsequent experiments. Preferred gene editing reagents are provided in the examples of the present invention.

As another alternative, the method for down-regulating the expression of FLO18 protein in plants may include: (1) transferring an interfering molecule that interferes with FLO18 gene expression into plant cells, tissues, organs or seeds, and obtaining the interfering molecule transformed into the interfering molecule. (2) regenerate the plant cell, tissue, organ or seed obtained in step (1) into the interfering molecule into a plant. Preferably, the method further comprises: (3) selecting the plant cells, tissues or organs into which the vector has been transferred; and (4) regenerating the plant cells, tissues or organs in step (3) into plants.

The methods can be carried out using any suitable conventional means, including reagents, temperature, pressure conditions, and the like.

Plant-directed screening and molecular markers

Based on the new findings of the present inventors, the present invention provides a molecular marker suitable for identifying plants with high yield, high endosperm quality or good starch development, namely the FLO18 gene. The present invention also relates to specific molecular markers designed for the FLO18 gene, and identification strategies.

Therefore, the present invention provides a method for targeted selection or identification of plants whose agronomic traits are regulated, comprising: identifying the expression or activity of FLO18 protein in a test plant, if the expression or activity of FLO18 protein in the test plant is higher than that in this type of plant The average value of FLO18 protein expression or activity in (control plants), then it is a grass plant with high yield, full grain, good grain filling, good starch development, large starch granule diameter, or capable of forming horny endosperm; or, if the test The expression or activity of FLO18 protein in the plant is lower than the average value of the expression or activity of FLO18 protein in this type of plant (control plant), which means that the yield is low, the grain is not full, the grain filling is not ideal, the starch development is not ideal, and the starch granule is not good. A grassy plant with a small diameter or capable of forming a silty endosperm.

According to the new findings of the present invention, those skilled in the art can use any of the various techniques known in the art or being developed to analyze nucleic acid sequences, and these techniques can all be included in the present invention. Said methods include, but are not limited to, sequencing, PCR amplification, probe methods, hybridization methods, restriction enzyme digestion analysis methods, and allelic polymorphism analysis methods (such as melting curve methods) for nucleic acid sequence analysis. identification, etc.

The identification method of the present invention only needs to perform PCR reaction and/or agarose gel electrophoresis, and by judging the length of the corresponding PCR product, the phenotype or yield of the sample to be tested can be accurately and rapidly judged, the cost is low, and it is suitable for It is suitable for large-scale identification, and the required sample size is small. If desired, one skilled in the art can design primers that identify the molecular markers.

The method for obtaining the DNA of the sample to be tested is a technique well known to those skilled in the art, such as the traditional phenol/chloroform/isoamyl alcohol method, or some commercially available DNA extraction kits, which are the well known to those skilled in the art. The polymerase chain reaction (PCR) technology is well known to those skilled in the art, and its basic principle is a method of enzymatically synthesizing specific DNA fragments in vitro. The methods of the present invention can be carried out using conventional PCR techniques.

The invention has a good application prospect in the aspects of molecular design breeding and using genetic engineering technology to improve crop varieties.

After knowing the function of the FLO18 gene, it can be used as a molecular marker to conduct targeted screening of plants. Substances or potential substances that regulate yield, endosperm quality or starch development by modulating this mechanism can also be screened based on this new finding.

The present invention provides a method for screening substances (potential substances) that promote the improvement of grass plant traits. The trait improvement includes: high yield, high endosperm quality or good starch development; the method includes: (1) adding candidate substances into into a system expressing FLO18 protein; (2) testing the system to observe the expression or activity of FLO18 protein, if its expression or activity is increased, it indicates that the candidate substance is a substance that promotes the improvement of grass plant traits.

The methods for screening substances acting on the target by taking a protein or gene or a specific region on it as a target are well known to those skilled in the art, and these methods can be used in the present invention. The candidate substances can be selected from: peptides, polymeric peptides, peptidomimetics, non-peptide compounds, carbohydrates, lipids, antibodies or antibody fragments, ligands, small organic molecules, small inorganic molecules, nucleic acid sequences, and the like. Depending on the type of substances to be screened, it is clear to those skilled in the art how to select a suitable screening method.

The detection of protein-protein interactions and the strength of the interactions can be performed using a variety of techniques well known to those skilled in the art, such as GST sedimentation technology (GST-Pull Down), bimolecular fluorescence complementation assay, yeast two-hybrid system or immunocoagulation. Precipitation technology, etc.

After large-scale screening, a class of substances that specifically act on the FLO18 protein or its encoding gene and have a regulatory effect on the improvement of grass plant traits can be obtained.

The present invention will be further described below in conjunction with specific embodiments. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods that do not indicate specific conditions in the following examples are usually in accordance with conventional conditions such as those described in J. Sambrook et al., Molecular Cloning Experiment Guide, 3rd Edition, Science Press, 2002, or according to the conditions described by the manufacturer. the proposed conditions.

Materials and methods

1. Acquisition and investigation of mutant material XA80

For the mutant material XA80, the rice grains were dehulled after the seeds were harvested. The Japanese SATAKE seed discriminating instrument and the Wanshen seed inspection system measured the grain length, grain width, and grain thickness of rice for statistical analysis.

2. Scanning electron microscope to observe the surface of seeds

Sample preparation: Randomly selected fully mature rice grains were sonicated in pure water for 10 minutes, and dried in an oven at 42°C for three days.

Sample fixation: stick the double-sided tape on the appropriate position of the copper table, pick up the sample with tweezers, make sure that the observation side is up and stick to the glue on the copper table. Apply conductive glue to make sure the viewing surface is clean.

Coating: After the sample is processed, vacuum spray gold coating is carried out.

Observation: After the samples were prepared, they were observed using a scanning electron microscope of Shimadzu JSM-6360LV type.

3. Semi-thin slices

Place the material for histological observation into a centrifuge tube containing FAA fixative. Put the centrifuge tube on ice and vacuumize, fully infiltrate the fixative solution into the tissue, and fix the internal structure of the material. After all the tissues were submerged in the fixative, the FAA fixative was replaced again and stored at 4°C. After dehydration using graded concentrations of ethanol, the material was embedded in epoxy resin for semi-thin sectioning.

4. Escherichia coli transformation and Agrobacterium transformation

E. coli transformation: The competent DH5α E. coli competent cells stored in a -80°C refrigerator were placed on ice for 5 min. An appropriate amount of plasmid was added to the competent cells in a mixed state of ice and water, the bottom of the tube was gently poked, and it was allowed to stand on ice for 15 minutes. The competent cells were immediately placed on ice after heat shock at 42°C for 40 s. After 2 minutes, 500 μl of anti-LB liquid medium was added. The competent cells were incubated in a 37°C incubator for 1 hour, and the bacterial solution was spread on the LB solid medium containing the corresponding resistance. Incubate overnight at 37°C.

Agrobacterium transformation: The Agrobacterium competent cell EHA105 was taken out from the -80°C refrigerator and thawed on ice. Add 0.01-1 μg of plasmid to the competent cells, stir the bottom of the tube to mix. Follow the sequence of 5 min on ice, 5 min in liquid nitrogen, 5 min at 37°C, and 5 min on ice. Add 500 microliters of anti-YEP-free liquid medium and incubate at 28°C for 2 to 3 hours. 5000rpm, 1min centrifugation to enrich the colony, leaving 100 microliters of bacterial solution in the tube. The enriched bacterial solution was spread on the corresponding resistant YEP solid medium. 28°C incubator for 2 days.

5. Subcellular localization of FLO18

(1) Construction of FLO18-GFP fusion protein

In order to observe the cellular sublocalization of GLW7, the present inventors constructed a FLO18-GFP fusion protein. Protoplasts were chemically transformed after sequencing was verified to be correct.

Cultivation of rice protoplasts: After the rice seeds were sterilized and dried in the ultra-clean bench, the seeds were sown on 1/2 MS medium and cultivated at 30°C for 7-10 days. Cut off the roots and leaves from the rice seedlings, using the green tissue of the stems and leaf sheaths. Cut into 0.5mm pieces with a blade, and immediately transfer the cut pieces to 20-30ml of 0.6M D-mannitol in a 50ml centrifuge tube (wrapped in tin foil) in the dark for 10min.

The enzymatic hydrolysis solution was prepared, and the formula is shown in Table 1.

Table 1

The preparation process of the enzymatic hydrolyzate solution is as follows (take 20ml as an example, the specific dosage is based on the sample amount): take 1ml of 0.2M MES (pH5.7) solution and heat it to 70°C for 3-5 minutes; add 0.3g Cellulase RS, 0.15 g Macerozyme R-10, 15ml of 0.8M mannitol, solution at 55°C, 10min; cooled to room temperature (25°C), added 200ul 1M CaCl ₂ and 200ul 100mg/ml BSA respectively, added water to make up to 20ml; use a 40um syringe and Filter the enzyme solution into another 50ml centrifuge tube with a 0.45um filter.

Prepare a small Erlenmeyer flask wrapped in tin foil, remove the mannitol from step 4, and immerse the small segment in the enzyme solution. The conical flask can be vacuum filtered for 25min to fully infiltrate the rice material with the enzymatic hydrolyzate. Place on a shaker at room temperature of 25°C with a rotational speed of 60 for 4 hours. Add 20ml of W5 (the same volume as the enzymatic hydrolysis solution) into the digested conical flask, and shake vigorously for 10s. The preparation of W5 is shown in Table 2.

Table 2

Final concentration Mother liquor concentration 100ml 200ml NaCl 154mM (0.9%) 0.9g 1.8g CaCl₂ 125mM (1.84%) 1M 12.5ml 25ml anhydrous dextrose 5mM (0.1%) 0.09 0.18g KCl 5mM 1M 500ul 1ml MES(pH5.7) 2mM 0.2M 1ml 2ml

Filter the protoplasts with a 40um nylon mesh into a 50ml centrifuge tube, rinse the conical flask with W52 solution, equilibrate and centrifuge at 100×G, (ACC 8; DEC 5), centrifuge for two minutes, and aspirate the supernatant. Add 10ml of W5, mix gently, 100XG, (ACC8; DEC 5), centrifuge for two minutes, and aspirate the supernatant. Resuspend with 1 ml of MMG, transfer the protoplasts in the centrifuge tube to a 2 mL EP tube, and count on a hemocytometer. The MMG formula is shown in Table 3.

table 3

Mother liquor concentration Final concentration 2ml 5ml 8ml 10ml Mannitol 0.8M 0.5M 1250ul 3.125ml 5ml 6.25ml MgCl₂ 1M 15mM 30ul 75ul 120ul 150ul MES(pH5.7) 0.2M 4mM 40ul 100ul 160ul 200ul

100×G, (ACC 8; DEC 5), centrifuge for two minutes, aspirate the supernatant, and add the corresponding volume of MMG to resuspend the protoplasts according to the previous microscopic examination.

(2) PEG-mediated transfection

The formula of PEG solution is shown in Table 4.

Table 4

Mother liquor concentration Final concentration 5ml PEG4000 40%(W/V) 2g Mannitol 0.8M 0.2M 1.25ml

After the PEG solution is prepared, it should be placed in an oven at 65 °C to melt it, and it can be cooled to room temperature during use.

Transfection steps: ① Prepare a 2ML centrifuge tube, add 10ug DNA prepared before to the bottom of the tube (generally 100μL of protoplast plus 10ugDNA, for BiFC or co-expression system DNA content can reach 10ug and 15ug), add 100uL of protoplast, Do not mix for now. ②Add 110ul of the newly prepared PEG solution. When adding the PEG solution, put the PE tube slightly flat, and then slowly add it along the tube wall. After adding a sample of PEG, mix it gently immediately. ③ Place in the dark at room temperature for 10-20min. ④ Slowly add 440ul of W5 solution to stop the reaction, 100×G, increase 8 to 5 (ACC 8; DEC 5), centrifuge for 2 minutes; discard the supernatant. ⑤ Repeat step ④. ⑥Resuspend in 500ul W5 solution. ⑦Put it in a carton, put it in a 26℃ incubator, and cultivate for 6-16 hours.

6. Sequence information

In the FLO18 gene sequence, bases 1 to 126 are 5'UTRs, positions 4716 to 5171 are 3'UTRs, and the yellow is the protein coding region.

OsFLO18 (LOC_Os07g32390) gene sequence (SEQ ID NO: 3):

GATCCAACCACCACCACCTTCTTCTCCTCCTCTTCCGCTCGACGCCATCACCACGCACTTGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAAACCCTAAGCCTCGCCCGCTATCCAGCCAAGCACCG ATGGCGACGGACGCGAACCC GGAGGCGGCGGCGCCGCCGCCGCAGTTACTGGTGGACGAGGGCTACGAGTTCTGCGCGCCCAAGTTCTTCGACTTCG TCTGCGACGAGACGGAGGAGGAAATCCGCGCCGCCGAGCGCTGGTTCGAGGCCTCCGCCAGCCACGCCCCTTCCC GTACGCTCCCCAATCCCCACCCCGCCGCCTCGATCATCCCCCCCTCTCCCGTGTTTTGCTTTTTTTGCGCTGATTAGGGCTCTTCTTCTTTTACCTGCTTGCAG CGTTCGCTCCGAGGATCAAGGAGTCGAGGGCGGAGGTCAAGATCGAGAGCCT CTGCGACTTCACCGACGCGGAGCCGATTCCGAAGG TACGAAGCAGAAAGCTGCGGGAATAACCCCCCAATTTTGCCCTAATTTCTGTAAATCTCCCCCCTTCTTATTGTCCGTTGGCCTCTGCGTTCGTCATGAT AGGAGGTAGCAGTGGAGGA GGCAGCAGGAAGCGCCGCCAATCCCTCGCAGAATTCTGATGGG TAAGAGTTGGAGAACTAAAGAAATTGTTTATGATGATTCGTTGTGATCTGATGAAGTTAATCGATTTCTCGTGTGTTTTTCATCTTCACTTTTGGATGTTCA GGAATGTGC AACAGAATAAGGACGGCTCCATCAAACTTGT ACGTGATTTCTTGATGTTTTCATGTTTTCTATGTTCTCAGTTTCAGTAAGTACTAGTGTTGGGTTTCTAGCAGCTCACATCCTGAATTCTTCAGGT CCATGAAGCAAATCCCAGTGAAAATTG TGTCACTGACGGCGATCATAAGCACCAAGAAAG GTGCATTTCAAGACCTCAATTTGTTGGTTCATTCC GTTTCTTGCTGGTTTTGGCATGTGAATGACTAATTGGTTGTTTTGGGCAG TGATGCAATGTTGGAGTCGCCACCAGCAGAGGAGGA TGAGAAGGAATCGCCAAAATCCTTCGAGTTTGTCCCTTCCAATGCAAAAT GTAAGTTGCTAGTTATATATGAGCAATGGCAGTGGGGAAGAACTCTGATCCCTGGATTTTGATATCAATGTTGCATGGCTTTAG CAGCAGATGTTGCTTCTAGC ACACCGAAGATTCAGAGGCCTCCACCCGTCAAAGCTGTCACTACGGTGCCTACCTGTCCCAAGCTGACAGTGAAGAC AGAAGCCTTCACTCCGAAGGTGCAGGCAACGAACTCCTCCAGAGGTCTTGCACCCTTGACTGGCTCAAGGGCACATC CATCTGCTTTGAAGCAGTCGATGAGCGTCAAGAGGAGTGTGATCAA GTAAGAGTCATGCTCACTCATAAAAAAATATATTAATCTGGAGATTATTTTCTTACTAATGTCTGAATTGTTGGTTTTTCAG ATGCCCTCGTGAGTTGCTGGCTGGGA AGGCCGCTACTGCTGCGAATGAAATCGCACAAGAAAATCAAGCTGTCAAGAGACAGAAGCTTGATGATGGGAGGACA AGACAG GTAATACTGGGATGAGATTAAAGATGGCACTTCAAATACATGTGTTTGCGTTGCGCGAGATTTCTGAGAAACATTGTGTTTGCCGTCGTGGTTAG ATACTGAACGTGAAGACAAGGACCCTGCCTCACAAGGGGAGAGGAGGTGGTCT AGCAGGAAGCACCGAGATGAGCCTGTCGGCCATGAGGAAGCACCGTGACGATTCACACTCTCTCAAGG TAGCTCCCAAGTTGCTCTTCTGACTCTTAAATGATAGACTTAATTGTAATGTTTCTTGTGAGAATGCAAGTATAATGTGATTTTGTTATTGTGAGCTTGGCACTTAGCAACTTTTGTTTCTGCCATGTGCAGG AAGT GACTCACTATATATCGGCAGCAGAGA TGGTAAAGAAGTTCGAGTCTGGGACTAGAGAGTTGGCCATCCCTCACAACAGATCGCTTTCTCATG TTAGCACTCATGTGCCCTTTATTATTAATTTTAAATTGTGTGCATTTCTCCAATTTTATTAATATACATGTCTTCTCAGTCTTGTTGTGGGTATTTGGTTCCATACAAAGACCGATGTCCTTTTTGTGATACTGATATCTGCATATCAAATATTGCACAGTTATTACATCTGAACCGTTGACGACGAAATCATTATTGGTACTAAGAATTTTGACATATTGCAATTTGTGCACAATGCAGG A GGATGCAGCAACCGCATTACAAAGAAGAACAAAGCTGATGCTAACAAGGCCAAAGGAACCTGAGTTCCAGACCTCCC ACAGGGTCCGTGCTGTCAGAGTGAAAAGCTCTGCTGAGCTAGAGGAGGAGATGCTAGCCAAAATTCCGAAGTTCAGA GCACGGCCGTTTAACAAAAAA GTGAGAAATTTGTTTGCTTTGGTTTTGATTATACTAACTACTCAATCCAGGATCTGAAGACTAACTATTTGTTTGCTTCTCCTTTCAG ATTGCGGAGGCCCCTTCATTTCCTCCTCTTCCAAGGAAAGCTCCA CAGCTTCCTGAATTTAATG TACGTCTTGTTTGGCTGATTCAGAAATTTATGAGCTCCACAGCTTTGTTTGGCTGATTCAGTTATTCCTTTCCAACACCTACAGG AGTTTCATCTTAAAACGATGGAGAGAGCTACACGACATGCAGACACCTGT TCCGAAGCTTCTTCCGTGGGGACTATCAGG GTAAGCTCCCTTCACTTAACCTGGTTGCTAATATCAGTATGTAAATTCTTCTTCTTATTAGTCTCCTAAAAATTAAAATTCTTGGCATTACTGAAAGTTTAATCAAATCTTGCAG AGTCAGAGC AGTAAGCCACTGACCTTGACAGCGCCAAAACCACC CCAACTTGAGACAGCATTACGAGCTAGACCACCAAGG TTTCCCTTTGCCACTTGCATCATTCAGAAAACAAGCCATTTTATGTTATTGATTATTTTCGATTACTCTAACATCATCTTTCTGCAGG GTGAAAAGTTCTCAGGAATTGGAACTAGAAGAATTAGAAAAGGCTCCCAAGTTCAAGGCAAAGCCACTGAA CAAAAAG GTCTCCTTATCTTCTTGAAGCTTGTACATTTGATACATTGATTGCATTGTGCTTCAAAACATCCAGTCTGAACATCATTTCTTGCAG ATTCTCGAGAGCAAGGGTGATATTGGTGTGTTTCCACATCTAAAGGCTCAACCAACAGCC CCCAAGGAATTCCATTTCTCTACCGATGATCGCTTGGGTCCTCCTGCAGTTGTGGATCTATTTGATAAG GTGAGGGTTCTAAGTCATGCTAGTAAGCTCATTATTTTTTTTCCAGAAACCTTTCTTCTAAGTCCTTTTCTATTTTGTAG CTCTC TCTTTGCTCAGAATCTTCATATCATAGCAAGAAAGATGTGCCCAGATTGACAATACCAAACCCCTTCAATTTACATA CAGAT GTAAGCAGTATGAAAGTTATTTGTTTTCCCAACACATTGCATTTACCTATCTGTTGCAGGCAAATAACTTTGTTATCCATGTTACAG GAAAGAGGGCATGAGAAAGAGAGGCAGCTAGCTGCACAACTGCTGCAAAAGCAATTACAGGA AGAGAAAGCCAGGATTCCGAAGGCTAATCCTTATCCCTACACAACTGACTATCCAGT GGTACGTGCAAAGTAACATACCGAATATTACATGTCCTGGTTGCTGACCATCTCATATTCTGCTAGCCTTAACATTTTCTCTCTCTTCA GATACCAC CGAAGCCTGAACCAAAACCATGCACGAGGCCAGAAGGCTTTCAGTTAGAGAGTTTGGTGAGGCATGAGATGGAGCAA CAGAGGATAATGGAAGA AAGAGAGAGGATGGAGAGGGAAGAGGCCCAGAGGAGAGTAGTGAAGGCACACCCCATAAT GAAAGAG TAAGCCTTCACATTACGGTTTCTCTTGTCCCAACAGTTCCTGGTTTCCCTTATAAGATGCATATTAATACTGTTAATTTTGTTGCAG GGATCCCATTCCTCTTCCAGAGAAAGAGAGGAAGCCTCTCACTGAAGTTCAACCACTTAA GTTACATGTTGATGAAAGGGCGGTCCAAAGATCAGAATTTGACAATATGGT CAGCAAAGCAAGCTTGTTCTATTATCTGGTTTGGAGGAGCCTGCCAAATTGGCTGAATTTACTGAGCAATTCGTTGTCGTGCAGGT GAAGGAAAAGGAAATAA CTTACAAGAGATTGCGAGAGGAAAATGAATTCGCACAAAAG GTTATTATTTTCTTATGCCTTCTTCTCAAAATCCCTACATTGTTGGATGCATTTTCATCTGTTCTGGCAACTAACTTGCTGTGATCTTTTCATGTGAAG ATTGAGGAAGAGAA AGCGTTGAAGCAGCTCAGGAGGACCTTGGTGCCACAAGCACGGCCTCTCCCGAAGTTTGACAGACCATTTCGTCCCC AGAG GTAAGAGCAGAAACGGCCAAGATTTTTGCCTACAATGTCATCGACCTTTCCGTTTCATCTCACAAACGTGAACTCGTTGTTTCGCACACAG ATCAACCAAGCAGGTGACGAGGCCGAAGTCCCCACAGCTTCAGGTCGACCAAAGAGGGG CAAGAAGGCACGCCTTCATCAGATGA TCCAACTCTCCGGCTCATCTTGTTGTCGTCTATCGGTTACCCACTGGCTGCTGCTGTTTTCTCTTCTGTTCTTGACGTCAAGATCATCCCCAATTCTCGCAGCGATTCTTTCTGACCAATACATAAATAGGCCGATCCAATCCTGTTATATAGTCAGTCAACACAGCTGACGGCGTGTGTATGCTCCTTTAGGGTTCTGCTGATGC TTGACTGCATTATTGTAAATTCAGTTTGTGTGCTGTGTGTGTGTGCTGTTCGATGAACGACAATAGGATGACACCCTGTCAGTGTCCTCCTCGTCTGTTTCGACGAATGCAAGTTGTATTCATTATTTGTTTATCATCTCTGAACTATCCTGGAGTTCTGTCGATGTTGAATAATCTAATTCATATCTCGGCACTTGGAAAACTCTCGTGAGAAACATATGTACCAAGAAAAACTGATTCATGTCAACAA

OsFLO18 CDS sequence (SEQ ID NO: 1):

ATGGCGACGGACGCGAACCCGGAGGCGGCGGCGCCGCCGCCGCAGTTACTGGTGGACGAGGGCTACGAGTTCTGCGCGCCCAAGTTCTTCGACTTCGTCTGCGACGAGACGGAGGAGGAAATCCGCGCCGCCGAGCGCTGGTTCGAGGCCTCCGCCAGCCACGCCCCTTCCCCGTTCGCTCCGAGGATCAAGGAGTCGAGGGCGGAGGTCAAGATCGAGAGCCTCTGCGACTTCACCGACGCGGAGCCGATTCCGAAGGAGGTAGCAGTGGAGGAGGCAGCAGGAAGCGCCGCCAATCCCTCGCAGAATTCTGATGGGAATGTGCAACAGAATAAGGACGGCTCCATCAAACTTGTCCATGAAGCAAATCCCAGTGAAAATTGTGTCACTGACGGCGATCATAAGCACCAAGAAAGTGATGCAATGTTGGAGTCGCCACCAGCAGAGGAGGATGAGAAGGAATCGCCAAAATCCTTCGAGTTTGTCCCTTCCAATGCAAAATCAGCAGATGTTGCTTCTAGCACACCGAAGATTCAGAGGCCTCCACCCGTCAAAGCTGTCACTACGGTGCCTACCTGTCCCAAGCTGACAGTGAAGACAGAAGCCTTCACTCCGAAGGTGCAGGCAACGAACTCCTCCAGAGGTCTTGCACCCTTGACTGGCTCAAGGGCACATCCATCTGCTTTGAAGCAGTCGATGAGCGTCAAGAGGAGTGTGATCAAATGCCCTCGTGAGTTGCTGGCTGGGAAGGCCGCTACTGCTGCGAATGAAATCGCACAAGAAAATCAAGCTGTCAAGAGACAGAAGCTTGATGATGGGAGGACAAGACAGATACTGAACGTGAAGACAAGGACCCTGCCTCACAAGGGGAGAGGAGGTGGTCTAGCAGGAAGCACCGAGATGAGCCTGTCGGCCATGAGGAAGCACCGTGACGATTCACACTCTCTCAAGGAAGTGACTCACTATATATCGGCAGCAGAGATGGTAAAGAAGTTCGAGT CTGGGACTAGAGAGTTGGCCATCCCTCACAACAGATCGCTTTCTCATGAGGATGCAGCAACCGCATTACAAAGAAGAACAAAGCTGATGCTAACAAGGCCAAAGGAACCTGAGTTCCAGACCTCCCACAGGGTCCGTGCTGTCAGAGTGAAAAGCTCTGCTGAGCTAGAGGAGGAGATGCTAGCCAAAATTCCGAAGTTCAGAGCACGGCCGTTTAACAAAAAAATTGCGGAGGCCCCTTCATTTCCTCCTCTTCCAAGGAAAGCTCCACAGCTTCCTGAATTTAATGAGTTTCATCTTAAAACGATGGAGAGAGCTACACGACATGCAGACACCTGTTCCGAAGCTTCTTCCGTGGGGACTATCAGGAGTCAGAGCAGTAAGCCACTGACCTTGACAGCGCCAAAACCACCCCAACTTGAGACAGCATTACGAGCTAGACCACCAAGGGTGAAAAGTTCTCAGGAATTGGAACTAGAAGAATTAGAAAAGGCTCCCAAGTTCAAGGCAAAGCCACTGAACAAAAAGATTCTCGAGAGCAAGGGTGATATTGGTGTGTTTCCACATCTAAAGGCTCAACCAACAGCCCCCAAGGAATTCCATTTCTCTACCGATGATCGCTTGGGTCCTCCTGCAGTTGTGGATCTATTTGATAAGCTCTCTCTTTGCTCAGAATCTTCATATCATAGCAAGAAAGATGTGCCCAGATTGACAATACCAAACCCCTTCAATTTACATACAGATGAAAGAGGGCATGAGAAAGAGAGGCAGCTAGCTGCACAACTGCTGCAAAAGCAATTACAGGAAGAGAAAGCCAGGATTCCGAAGGCTAATCCTTATCCCTACACAACTGACTATCCAGTGATACCACCGAAGCCTGAACCAAAACCATGCACGAGGCCAGAAGGCTTTCAGTTAGAGAGTTTGGTGAGGCATGAGATGGAGCAACAGAGGATAATGGAAGAAAGAGAGAGGATGGAGAGGGAAGAGGCCCAGAGGAG AGTAGTGAAGGCACACCCCATAATGAAAGAGGATCCCATTCCTCTTCCAGAGAAAGAGAGGAAGCCTCTCACTGAAGTTCAACCACTTAAGTTACATGTTGATGAAAGGGCGGTCCAAAGATCAGAATTTGACAATATGGTGAAGGAAAAGGAAATAACTTACAAGAGATTGCGAGAGGAAAATGAATTCGCACAAAAGATTGAGGAAGAGAAAGCGTTGAAGCAGCTCAGGAGGACCTTGGTGCCACAAGCACGGCCTCTCCCGAAGTTTGACAGACCATTTCGTCCCCAGAGATCAACCAAGCAGGTGACGAGGCCGAAGTCCCCACAGCTTCAGGTCGACCAAAGAGGGGCAAGAAGGCACGCCTTCATCAGATGA

OsFLO18 protein sequence (SEQ ID NO:2)

MATDANPEAAAPPPQLLVDEGYEFCAPKFFDFVCDETEEEIRAAERWFEASASHAPSPFAPRIKESRAEVKIESLCDFTDAEPIPKEVAVEEAAGSAANPSQNSDGNVQQNKDGSIKLVHEANPSENCVTDGDHKHQESDAMLESPPAEEDEKESPKSFEFVPSNAKSADVASSTPKIQRPPPVKAVTTVPTCPKLTVKTEAFTPKVQATNSSRGLAPLTGSRAHPSALKQSMSVKRSVIKCPRELLAGKAATAANEIAQENQAVKRQKLDDGRTRQILNVKTRTLPHKGRGGGLAGSTEMSLSAMRKHRDDSHSLKEVTHYISAAEMVKKFESGTRELAIPHNRSLSHEDAATALQRRTKLMLTRPKEPEFQTSHRVRAVRVKSSAELEEEMLAKIPKFRARPFNKKIAEAPSFPPLPRKAPQLPEFNEFHLKTMERATRHADTCSEASSVGTIRSQSSKPLTLTAPKPPQLETALRARPPRVKSSQELELEELEKAPKFKAKPLNKKILESKGDIGVFPHLKAQPTAPKEFHFSTDDRLGPPAVVDLFDKLSLCSESSYHSKKDVPRLTIPNPFNLHTDERGHEKERQLAAQLLQKQLQEEKARIPKANPYPYTTDYPVIPPKPEPKPCTRPEGFQLESLVRHEMEQQRIMEERERMEREEAQRRVVKAHPIMKEDPIPLPEKERKPLTEVQPLKLHVDERAVQRSEFDNMVKEKEITYKRLREENEFAQKIEEEKALKQLRRTLVPQARPLPKFDRPFRPQRSTKQVTRPKSPQLQVDQRGARRHAFIR

Amplification primers used to amplify the full-length OsFLO18 gene:

PrimerF: GATCCAACCACCACCACCTTCTTCTCCTCCT (SEQ ID NO: 4);

PrimerR: TTGTTGACATGAATCAGTTTTTCTTGGTAC (SEQ ID NO: 5).

Example 1. Candidate gene analysis

The inventors obtained a stably inherited farinaceous endosperm mutant line using japonica IL-9 (Tohoku IL9) mutagenized by γ-rays, which the inventors named XA80.

The inventors used the mutant material XA80 to construct a targeting population by crossing with 9311 (Fig. 1a). The gene was located in the range of 19.25M to 19.26M on rice chromosome 7 by map-based cloning, the Nipponbare sequence at the corresponding position was searched through the RAP-DB database, primers were designed to amplify the covering fragment, and the mutant and wild Through the sequence detection in this region, the inventors found that there is a single-base mutation from T to A in the mutant sequence (Fig. 1b). This mutation is located exactly on exon 18 of LOC_Os07g32390. The inventors identified the LOC_Os07g32390 gene as a candidate gene, named as the FLO18 gene.

The FLO18 gene encodes a microtubule-binding protein, FLO18. The full length is 5171 bases. In the mutant plant (XA80), the mutation of the base T to A in exon 18 changes the amino acid encoding lysine (AAG) into a stop codon (TAG), resulting in a deletion of 195 amino acids, Loss of gene function. The gene spatiotemporal expression profiles of FLO18 were compared in the RiceXPro (https://ricexpro.dna.affrc.go.jp/) database, and it was found that the FLO18 gene was specifically expressed during the reproductive growth stage of rice, which was consistent with the plant phenotype.

For the FLO18 gene, the inventors conducted a homology analysis in rice, and used the FLO18 protein sequence to obtain the protein sequence of 9 genes with the same structural and functional domain as FLO18 on the MSU website database, belonging to the TPX2 protein family. Some genes have multiple transcripts translated into multiple proteins. Subsequently, MEGA was used for cluster analysis, and the Neightbor-joining method was used to construct a phylogenetic tree. The Bootstrap value was set to 2000 (as shown in Figure 1c). It can be seen from the results that the TPX2 protein is divided into two branches in rice, and the TPX2 family has certain functional conservation.

Example 2. Gene mapping population construction and phenotypic investigation

In this example, the inventors conducted a phenotypic investigation on XA80 and compared it with wild-type plants.

Through phenotypic investigation, the inventors found that compared with the wild-type plant, the mutant has no significant difference in plant height, plant type and heading stage. Phenotypic photographs of representative plants are shown in Figure 2a, with the wild type on the left and the mutant on the right.

The inventors obtained the grains of the mutant and wild-type plants, and compared them after removing the glumes. The results showed that there were obvious differences in the size of grains. The wild-type grains were transparent and uniform (the upper left panel of Fig. 2b), while the mutants had opaque endosperm (the lower left panel of Fig. 2b), and the kernels were smaller. The mutant and wild-type grains observed under the asana microscope, the wild-type grains are plump and smooth, the cross-section is transparent and crystal-clear and compact (the upper right picture of Figure 2b), which is horny endosperm; the mutant grains are shrunken and small in shape. The surface is rough, loose and powdery white, and is a silty endosperm (bottom right of Figure 2b).

The thousand-kernel weight of the wild type was 19.6835±0.211 g, and the thousand-kernel weight of the mutant was 15.9428±1.551 g. The thousand-kernel weight of the mutant was 19% less than that of the wild type, p-Value=0.004147, and the thousand-kernel weight of the mutant was significantly lower than that of the wild type. (Fig. 2c, left panel).

After rice flowering, every 3 days from the first day, the dry weight of grains with glumes in wild-type and mutant rice was measured, and the rice grain filling rate was plotted (Fig. 2c, right panel). It can be seen from the results that the grain filling rate of the mutants was not significant compared with the normal individuals in the first 3 days, and the dry matter accumulation gradually slowed down from the 6th day (ie: the grain filling rate decreased). The dry weight after final maturity was significantly lower than that of the wild type.

The dehulled grains were compared for statistics and grain shape (Fig. 2d). The grain length of the wild type after shelling was 4.8614±0.044cm, and the grain length of the mutant after shelling was 4.8771±0.040cm, p-Value=0.7625, there was no difference between the two. After shelling, the grain width of the wild type was 2.4604±0.027cm, and that of the mutant was 2.3099±0.040cm, p-Value=3.716e-11, and the grain width of the mutant was significantly smaller than that of the wild type. The grain thickness of the wild type after shelling was 1.8923±0.016cm, and the grain thickness of the mutant after shelling was 1.5811±0.038cm, p-Value<2.2e-16, and the grain thickness of the mutant was significantly smaller than that of the wild type.

The seeds were observed under a scanning electron microscope. From the perspective of the whole seed, the mutant seeds were shriveled. In addition to the edges along the vertical axis, there were also edges along the horizontal axis. From the cross section, the wild type was full of filling and the fracture surface was neat. . The mutants had incomplete grain filling, rough fracture surfaces, and gaps between the seed coat and the endosperm. Judging from the state of starch granules, the wild-type starch granules were well developed and were regular crystalline, and the starch granules were closely arranged. The starch in the mutant was not fully developed, and the starch granules were obviously smaller, and the starch granules were spherical, and the space between the starch granules was larger (Fig. 2e).

The cellular structure of endosperm was observed under light microscope and transmission electron microscope by sampling 2 days after flowering (2DAF), 4DAF, 6DAF, 8DAF, 10DAF, 12DAF. Through semi-thin sectioning, the inventors did not observe obvious difference in cell structure between the mutant and the wild type, but found that there was a certain difference in the depth of staining. Observation under transmission electron microscope showed that the development of amyloid in the mutant was significantly slower than that of the wild type, and the crystal structure was loose and the granules were small; and the existence of protein bodies was rarely observed (Fig. 2f, g).

Example 3. Establishment of replenishing plants

The inventors used the genome of the IL9 wild-type plant as a template, and amplified with the following gene complementary sequence amplification primers to obtain a sequence (8619bp) containing the FLO18 gene, its promoter and upstream and downstream partial regions (excluding coding regions), It was cloned into the pCAMBIA1300 vector backbone to construct a complementary vector FLO18-Complementary (FLO18-CP). After that, the inventors transformed the target vector by inducing the mature embryos of the mutant XA80 to differentiate into callus, using the method of Agrobacterium transformation, and obtained transgenic plants through a series of steps of co-cultivation, screening, pre-differentiation, differentiation and rooting . The transgenic plants were detected using hygromycin primers and mutation site-specific primers, and transgenic positive plants were screened. Phenotypes were confirmed at T0 and T1 generations.

Gene complementary sequence amplification primers:

PrimerF: ATGTGTTCTTAATTGTGGGGATCAGAATTT (SEQ ID NO: 6);

PrimerR: TTAGAAATCTCTTTGTTGCTAGCTGTTCTC (SEQ ID NO: 7).

The phenotype of the obtained T1 complemented plants was observed. The results showed that the XA80 mutant complemented by FLO18 showed the phenotype of japonica IL-9 wild-type, with plump and smooth grain shape, transparent and crystal-clear cross-section; its thousand-grain weight was also similar to that of wild-type plants. same.

Example 4. FLO18 is related to cell division and regulates endosperm quality

According to the aforementioned analysis, the present inventors considered that the loss of function of FLO18 resulted in unequal division of cells or chromosomes in the endosperm of mutant plants during cell division.

In order to observe whether this phenomenon exists, the inventors selected the endosperm of XA80 and IL9 with different fertilization days. The material was fixed and observed under a transmission electron microscope by ultrathin sectioning. The present inventors found that in the endosperm of rice 6DAP (6 days after pollination), nuclei were observed in cells surrounding vascular bundles. The left side of the figure is the normal individual, the right side is the mutant (as shown in Figure 3a), and the circles are the marked nuclei. The endosperm nuclei in normal individuals are dark gray and uniform in size; however, the endosperm nuclei in mutants vary in size, some of which are similar in size to normal individuals, while others are significantly smaller. This indicated that during the development of endosperm, the mutant did have uneven division of nuclei.

Therefore, FLO18 is associated with cell division and thus regulates endosperm quality.

Example 5. Subcellular localization of FLO18

The full-length CDS of FLO18 was amplified and connected upstream of the PA7-GFP vector to construct a PA7-GFP fusion protein. The fusion protein was transformed into rice protoplasts for transient expression and co-transformed into the mcherry-TUB6 vector. Observed under a Zeiss LSM880 laser confocal microscope. The results showed that FLO18 was abundantly expressed mainly in the cytoplasm, with no expression on the cell membrane and large central vacuole (Fig. 3b).

mcherry-TUB6 is mainly used to label tubulin, and the co-localization of FLO18 and TUB6 was found by co-transformation experiments, indicating that FLO18 interacts with tubulin (Figure 3c).

Summarize

The inventors cloned the gene FLO18, which plays an important role in the development of rice endosperm. It can control the division and development of endosperm cells, affect the development of the entire endosperm structure, and further affect the accumulation of storage substances, thereby affecting the quality of the endosperm. The yield of the FLO18 mutant was reduced but had no significant effect on the number of grains per panicle, panicle length and panicle shape. In traditional quality breeding, with the improvement of quality, the yield will inevitably decline. The research on this gene will help those in the art to better understand and analyze the role of the cytoskeleton in the development of endosperm, and provide a new way for high-yield and high-quality breeding of rice and other plants.

All documents mentioned herein are incorporated by reference in this application as if each document were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

sequence listing

<110> Center for Excellence and Innovation in Molecular Plant Science, Chinese Academy of Sciences

<120> Gene FLO18 regulating endosperm development and quality

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Glu Ser Arg Ala Glu Val Lys Ile Glu Ser Leu Cys Asp Phe Thr Asp

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Ala Glu Pro Ile Pro Lys Glu Val Ala Val Glu Glu Ala Ala Gly Ser

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Asp Gly Ser Ile Lys Leu Val His Glu Ala Asn Pro Ser Glu Asn Cys

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Val Thr Asp Gly Asp His Lys His Gln Glu Ser Asp Ala Met Leu Glu

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Ser Pro Pro Ala Glu Glu Asp Glu Lys Glu Ser Pro Lys Ser Phe Glu

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Phe Val Pro Ser Asn Ala Lys Ser Ala Asp Val Ala Ser Ser Thr Pro

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Lys Ile Gln Arg Pro Pro Pro Val Lys Ala Val Thr Thr Val Pro Thr

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Cys Pro Lys Leu Thr Val Lys Thr Glu Ala Phe Thr Pro Lys Val Gln

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Ala Thr Asn Ser Ser Arg Gly Leu Ala Pro Leu Thr Gly Ser Arg Ala

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His Pro Ser Ala Leu Lys Gln Ser Met Ser Val Lys Arg Ser Val Ile

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Gly Arg Thr Arg Gln Ile Leu Asn Val Lys Thr Arg Thr Leu Pro His

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Lys Gly Arg Gly Gly Gly Leu Ala Gly Ser Thr Glu Met Ser Leu Ser

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Ala Pro Lys Glu Phe His Phe Ser Thr Asp Asp Arg Leu Gly Pro Pro

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tggctgattc agaaatttat gagctccaca gctttgtttg gctgattcag ttattccttt 2700

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ctgttccgaa gcttcttccg tggggactat cagggtaagc tcccttcact taacctggtt 2820

gctaatatca gtatgtaaat tcttcttctt attagtctcc taaaaattaa aattcttggc 2880

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acatcatctt tctgcagggt gaaaagttct caggaattgg aactagaaga attagaaaag 3120

gctcccaagt tcaaggcaaa gccactgaac aaaaaggtct ccttatcttc ttgaagcttg 3180

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cagattctcg agagcaaggg tgatattggt gtgtttccac atctaaaggc tcaaccaaca 3300

gcccccaagg aattccattt ctctaccgat gatcgcttgg gtcctcctgc agttgtggat 3360

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atagcaagaa agatgtgccc agattgacaa taccaaaccc cttcaattta catacagatg 3540

taagcagtat gaaagttatt tgttttccca acacattgca tttacctatc tgttgcaggc 3600

aaataacttt gttatccatg ttacaggaaa gagggcatga gaaagagagg cagctagctg 3660

cacaactgct gcaaaagcaa ttacaggaag agaaagccag gattccgaag gctaatcctt 3720

atccctacac aactgactat ccagtggtac gtgcaaagta acataccgaa tattacatgt 3780

cctggttgct gaccatctca tattctgcta gccttaacat tttctctctc ttcagatacc 3840

accgaagcct gaaccaaaac catgcacgag gccagaaggc tttcagttag agagtttggt 3900

gaggcatgag atggagcaac agaggataat ggaagaaaga gagaggatgg agagggaaga 3960

ggcccagagg agagtagtga aggcacaccc cataatgaaa gagtaagcct tcacattacg 4020

gtttctcttg tcccaacagt tcctggtttc ccttataaga tgcatattaa tactgttaat 4080

tttgttgcag ggatcccatt cctcttccag agaaagagag gaagcctctc actgaagttc 4140

aaccacttaa gttacatgtt gatgaaaggg cggtccaaag atcagaattt gacaatatgg 4200

tcagcaaagc aagcttgttc tattatctgg tttggaggag cctgccaaat tggctgaatt 4260

tactgagcaa ttcgttgtcg tgcaggtgaa ggaaaaggaa ataacttaca agagattgcg 4320

agaggaaaat gaattcgcac aaaaggttat tattttctta tgccttcttc tcaaaatccc 4380

tacattgttg gatgcatttt catctgttct ggcaactaac ttgctgtgat cttttcatgt 4440

gaagattgag gaagagaaag cgttgaagca gctcaggagg accttggtgc cacaagcacg 4500

gcctctcccg aagtttgaca gaccatttcg tccccagagg taagagcaga aacggccaag 4560

atttttgcct acaatgtcat cgacctttcc gtttcatctc acaaacgtga actcgttgtt 4620

tcgcacacag atcaaccaag caggtgacga ggccgaagtc cccacagctt caggtcgacc 4680

aaagaggggc aagaaggcac gccttcatca gatgatccaa ctctccggct catcttgttg 4740

tcgtctatcg gttacccact ggctgctgct gttttctctt ctgttcttga cgtcaagatc 4800

atccccaatt ctcgcagcga ttctttctga ccaatacata aataggccga tccaatcctg 4860

ttatatagtc agtcaacaca gctgacggcg tgtgtatgct cctttagggt tctgctgatg 4920

cttgactgca ttattgtaaa ttcagtttgt gtgctgtgtg tgtgtgctgt tcgatgaacg 4980

acaataggat gacaccctgt cagtgtcctc ctcgtctgtt tcgacgaatg caagttgtat 5040

tcattatttg tttatcatct ctgaactatc ctggagttct gtcgatgttg aataatctaa 5100

ttcatatctc ggcacttgga aaactctcgt gagaaacata tgtaccaaga aaaactgatt 5160

catgtcaaca a 5171

<210> 4

<211> 31

<212> DNA

<213> Primer

<400> 4

gatccaacca ccaccacctt cttctcctcc t 31

<210> 5

<211> 30

<212> DNA

<213> Primer

<400> 5

ttgttgacat gaatcagttt ttcttggtac 30

<210> 6

<211> 30

<212> DNA

<213> Primer

<400> 6

atgtgttctt aattgtgggg atcagaattt 30

<210> 7

<211> 30

<212> DNA

<213> Primer

<400> 7

ttagaaatct ctttgttgct agctgttctc 30

Claims (15)

1. Use of a FLO18 protein or a regulatory molecule thereof for regulating yield traits, endosperm traits or starch development traits of grasses. 2. purposes as claimed in claim 1, is characterized in that, described regulatory molecule is up-regulated molecule, the purposes of described FLO18 protein or its up-regulated molecule comprises being selected from: (i) increasing yield; preferably including increasing grain width or grain thickness, or promoting grain filling; (ii) improving the quality of the endosperm; preferably including promoting equal division of cells during endosperm development, or promoting the formation of horny endosperm; (iii) promoting starch development; preferably including increasing the diameter of starch granules. 3. purposes as claimed in claim 2 is characterized in that, described up-regulated molecule comprises: the expression cassette or expression construct that overexpresses described FLO18 protein; Or, the expression cassette or expression that improves described FLO18 protein translation efficiency construct; or, an up-regulated molecule that interacts with the FLO18 protein to increase its expression or activity. 4. purposes as claimed in claim 1 is characterized in that, described regulatory molecule is down-regulated molecule, and its purposes comprise being selected from: (i) reducing yield; preferably including reducing grain width or grain thickness, or reducing grain filling; (ii) reducing the quality of the endosperm; preferably including inhibiting the equal division of cells during endosperm development, or promoting the formation of a powdery endosperm; (iii) interfering with starch development; preferably including reducing starch granule diameter. 5. purposes as claimed in claim 4 is characterized in that, described down regulation molecule comprises: the reagent that knocks out or silences the gene encoding FLO18 protein, the reagent that inhibits FLO18 protein activity; Preferably, described down regulation molecule comprises: A gene editing reagent, a homologous recombination reagent or a site-directed mutagenesis reagent directed against the gene encoding the FLO18 protein, the reagent mutates the FLO18 protein with loss of function; or, an interfering molecule that specifically interferes with the expression of the gene encoding the FLO18 protein. 6. A method for regulating yield traits, endosperm traits or starch development traits of grass plants, comprising: regulating the expression or activity of FLO18 protein in plants. 7. The method of claim 6, wherein the regulation is to increase yield, improve endosperm quality or promote starch development; the method comprises: up-regulating the expression or activity of FLO18 protein in plants. 8. The method of claim 7, wherein the regulation is to reduce yield, reduce endosperm quality or interfere with starch development; the method comprises: down-regulating the expression or activity of FLO18 protein in plants. 9. The method of claim 7 or 8, wherein the up-regulating expression or activity of FLO18 protein in plants comprises: overexpressing FLO18 protein in plants; or, with up-regulated molecules that interact with FLO18 protein is modulated to increase the expression or activity of the FLO18 protein; or The down-regulation of the expression or activity of the FLO18 protein in plants includes: knocking out or silencing the gene encoding the FLO18 protein in plants, or inhibiting the activity of the FLO18 protein; preferably, it includes: performing gene editing with the CRISPR system to knock out the FLO18 protein Knock out the encoding gene of FLO18 protein by homologous recombination method; silence by interfering molecules that specifically interfere with the expression of FLO18 protein encoding gene; or perform loss-of-function mutation of FLO18 protein. 10. as described in claim 1 or 6, it is characterized in that, described gramineous plant is the plant that expresses FLO18 protein or its homologue; Preferably, described gramineous plant comprises selected from: rice, barley, wheat , oats, rye, corn, sorghum, Brachypodium. 11. as described in claim 1 or 6, it is characterized in that, the amino acid sequence of the polypeptide of described FLO18 is selected from the following group: (i) the polypeptide with the amino acid sequence shown in SEQ ID NO:2; The amino acid sequence shown in SEQ ID NO: 2 is formed by the substitution, deletion or addition of one or several amino acid residues, and has (i) polypeptide function and is derived from (i) polypeptide; (iii) amino acid sequence and The homology of the amino acid sequence shown in SEQ ID NO: 2 is ≥85%, and the polypeptide has the function of regulating the traits; (iv) the active fragment of the polypeptide of the amino acid sequence shown in SEQ ID NO: 2; or (v) in SEQ ID NO: 2 The polypeptide of the amino acid sequence shown in ID NO: 2 is formed by adding a tag sequence or an enzyme cleavage site sequence to the N-terminus or C-terminus thereof, or adding a signal peptide sequence to its N-terminus. 12. Use of FLO18 protein or the gene encoding it as a molecular marker for identifying traits of grasses; the traits include: yield traits, endosperm traits or starch development traits. 13. A method for directed selection or identification of high yield, high endosperm quality or good starch development, comprising: identification of the expression or activity of FLO18 protein in the test plant, if the expression or activity of FLO18 protein in the test plant is high If it is equal to or equal to the average value of FLO18 protein expression or activity in such plants, it is a grass plant with high yield, high endosperm quality or good starch development. 14. A method for screening a substance that promotes the improvement of grass plant traits, the trait improvement comprising: high yield, high endosperm quality or good starch development; the method comprises: (1) adding candidate substances to FLO18 protein-expressing plants; (2) Detecting the system, and observing the expression or activity of the FLO18 protein. If the expression or activity is increased, it indicates that the candidate substance is a substance that promotes the improvement of grass plant traits. 15. A grass cell, tissue or organ, wherein the up-regulated molecule of exogenous FLO18 protein is contained, and the up-regulated molecule comprises selected from: an expression cassette or an expression construct that overexpresses the FLO18 protein; or increases the An expression cassette or expression construct for FLO18 protein translation efficiency; or an up-regulated molecule that interacts with said FLO18 protein to increase its expression or activity.

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