Genes that regulate seed dormancy and germination in plants and their use

Abstract

The application provides a gene (GA2ox9) for regulating seed dormancy and germination of plants and application thereof. The GA2ox9 affects alpha amylase activity and soluble sugar content by regulating the content of active gibberellin (GA) in endosperm, changes abscisic acid (ABA) signal in the embryo, and further regulates seed dormancy and plant ear germination. The GA2ox9 gene deletion leads to the decrease of seed dormancy and the occurrence of ear germination, and the overexpression of the GA2ox9 gene improves seed dormancy. The application provides a new way for improving seed dormancy and reducing ear germination of plants.

Description

Technical Field

[0001] This invention belongs to the fields of botany and genetic engineering, and more specifically, this invention relates to genes that regulate dormancy and germination of plant seeds and their applications. Background Technology

[0002] With the acceleration of industrialization and informatization, and the improvement of human medical standards, the world's population is increasing rapidly. Food yield and superior germplasm resources are of paramount importance to people's livelihoods. Utilizing modern molecular genetics theories and methods to deeply study the molecular genetic mechanisms of crop yield formation, combined with molecular design breeding techniques, can help people better utilize high-quality germplasm resources.

[0003] Cereal crops are essential agricultural products, and how to effectively cultivate them on limited arable land has always been a focus of agricultural research. Studying methods to regulate crop cultivation is crucial. In particular, rice (Oryza sativa), a member of the Poaceae family, is one of the world's most important food crops, providing staple food for more than half of the global population. With the continued growth of the world's population, the decreasing arable land area, and the increasing environmental pollution, food production faces even more severe challenges. For some populous countries, high rice yields and quality directly impact the quality of life of the people, are of great significance for ensuring national welfare and people's livelihoods, and are also directly related to global food security.

[0004] In the breeding process, varieties with rapid and uniform seed germination, quick seedling emergence, and high economic yields are often selected for production. However, this selection method can also lead to some adverse phenomena. For example, high-yielding varieties often lose seed dormancy and exhibit strong pre-harvest sprouting during the harvest season when encountering hot and rainy weather, severely affecting rice yield and quality (Yang Jun et al., 1991). Pre-harvest sprouting (PHS) refers to the phenomenon of rice grains germinating on the panicle when encountering hot and humid conditions during the harvest period, and it is closely related to seed dormancy and germination. Pre-harvest sprouting not only causes reduced grain crop yield and decreased edible quality but also affects the quality of crop seed production. Statistics show that due to the frequent hot and rainy weather during the rice harvest season in southern China, pre-harvest sprouting occurs in about 6% of the sown area of ​​conventional rice and as much as 20% of the sown area of ​​hybrid rice.

[0005] The phenomenon of pre-harvest sprouting has been studied since the 1980s, and several QTLs related to pre-harvest sprouting have been identified, providing clues for the subsequent discovery and cloning of key genes. The regulation of pre-harvest sprouting is a complex physiological process influenced by numerous factors. In addition to external factors such as water, temperature, and light, it is also affected by hormones, sugars, reactive oxygen species, NO, and microRNAs within the seed.

[0006] Although the issue of spikelet germination has gradually gained attention, especially in the model plant Arabidopsis thaliana, the research foundation for its application in crops remains relatively weak. Summary of the Invention

[0007] The purpose of this invention is to provide genes that regulate plant seed dormancy and germination and their applications.

[0008] In a first aspect of the invention, a method for regulating seed dormancy and seed germination in grasses is provided, comprising: regulating the expression or activity of the GA2ox9 gene or its encoded protein in the plant, thereby regulating seed dormancy and seed germination.

[0009] In one or more embodiments, the method is selected from: (a) upregulating the expression or activity of the GA2ox9 gene or the protein encoded therein, thereby increasing seed dormancy or reducing germination (e.g., spike germination); or, (b) downregulating the expression or activity of the GA2ox9 gene or the protein encoded therein, thereby reducing plant seed dormancy or promoting germination (e.g., spike germination).

[0010] In one or more embodiments, the upregulation of the expression or activity of the GA2ox9 gene or the protein it encodes includes: transferring the GA2ox9 gene or an expression construct or vector containing the gene into a plant; or performing a gain-of-function mutation of the GA2ox9 gene in plants with low expression or activity of the GA2ox9 gene (including no expression or no activity).

[0011] In one or more embodiments, downregulating the expression or activity of the GA2ox9 gene or its encoded protein includes: knocking out or silencing the GA2ox9 gene in plants, or inhibiting the activity of the GA2ox9 protein; preferably, it includes: gene editing using a CRISPR system to knock out the GA2ox9 gene, silencing the GA2ox9 gene with an interfering molecule that specifically interferes with the expression of the GA2ox9 gene, knocking out the GA2ox9 gene by homologous recombination, or performing a loss-of-function mutation on the GA2ox9 gene; preferably, the downregulation method includes: modifying the GA2ox9 gene to cause a frameshift or premature occurrence of a terminator (the GA2ox9 protein it encodes provides termination); preferably, the modification targets positions 367-410 of the nucleotide sequence shown in SEQ ID NO:1 (preferably, positions 384-403 are the gRNA target binding region, and positions 381-383 are the PAM sequence); preferably, the nucleotide sequence of the sgRNA used in the gene editing is as shown in SEQ ID NO:3.

[0012] In one or more embodiments, the GA2ox9 gene or the protein it encodes increases seed dormancy or reduces germination by reducing the content of active gibberellins (preferably gibberellins in the seed endosperm), reducing α-amylase activity, reducing the content of soluble sugars, increasing abscisic acid (preferably abscisic acid in the embryo) sensitivity.

[0013] In one or more embodiments, downregulating the GA2ox9 gene or the protein it encodes increases the content of active gibberellins (preferably gibberellins in the seed endosperm), increases α-amylase activity, increases the content of soluble sugars, reduces abscisic acid (preferably abscisic acid in the embryo) sensitivity, reduces plant seed dormancy, or promotes germination.

[0014] In another aspect of the invention, the use of the GA2ox9 gene, the protein encoded therein, or regulators thereof is provided for regulating seed dormancy and seed germination in grasses.

[0015] In one or more embodiments, the regulator is the GA2ox9 gene or the protein it encodes is an expression construct or vector containing the GA2ox9 gene, used to improve seed dormancy or reduce germination (such as ear germination).

[0016] In one or more embodiments, the regulator is a downregulator of the GA2ox9 gene or its encoded protein, used to reduce plant seed dormancy or promote germination (e.g., spikelet germination).

[0017] In one or more embodiments, the downregulator includes: gene editing reagents that specifically edit the GA2ox9 gene, interfering RNA molecules (such as siRNA, shRNA) or antisense nucleotides that specifically interfere with the expression of the GA2ox9 gene, and reagents that knock out the GA2ox9 gene through homologous recombination.

[0018] In another aspect of the present invention, a method is provided for screening regulators that regulate seed dormancy and seed germination in grass plants, the method comprising: (1) adding a candidate substance to a system containing the GA2ox9 gene or its encoded protein; (2) detecting the system and observing the expression or activity of the GA2ox9 gene or its encoded protein; if the candidate substance upregulates (significantly upregulates, such as upregulating by 10%, 20%, 40%, 60%, 80%, 90% or higher) the GA2ox9 gene or its encoded protein, then the candidate substance is a regulator that increases seed dormancy or reduces germination; if the candidate substance downregulates (significantly downregulates, such as downregulating by 10%, 20%, 40%, 60%, 80%, 90% or higher) the expression or activity of the GA2ox9 gene or its encoded protein, then the candidate substance is a regulator that reduces plant seed dormancy or promotes germination.

[0019] In one or more embodiments, the screening further includes observing changes in the signaling pathway “GA2ox9-gibberellin-α-amylase-soluble sugar-abscisic acid” involved by the GA2ox9 gene or its encoded protein; if the candidate substance acts on the GA2ox9 gene or its encoded protein, thereby reducing the content of active gibberellin, reducing α-amylase activity, reducing the content of soluble sugar, or increasing abscisic acid (preferably embryo abscisic acid) sensitivity, it indicates that the candidate substance is a regulator that improves seed dormancy or reduces germination.

[0020] In one or more embodiments, the screening further includes observing changes in the signaling pathway “GA2ox9-gibberellin-α-amylase-soluble sugar-abscisic acid” involved by the GA2ox9 gene or its encoded protein; if the candidate substance acts on the GA2ox9 gene or its encoded protein, thereby increasing the content of active gibberellin, increasing α-amylase activity, increasing the content of soluble sugar or reducing abscisic acid sensitivity, it indicates that the candidate substance is a regulator that reduces plant seed dormancy or promotes germination.

[0021] In one or more embodiments, the system includes systems selected from: cell (culture) systems, subcellular (culture) systems, tissue (culture) systems, or animal systems.

[0022] In one or more embodiments, the candidate substances include (but are not limited to): regulatory molecules designed for the “GA2ox9-gibberellin-α-amylase-soluble sugar-abscisic acid” signaling pathway, or pathway proteins, or upstream or downstream proteins, genes or signaling pathways (such as but not limited to interfering molecules, nucleic acid inhibitors, binding molecules (such as antibodies or ligands)), CRISPR constructs, small molecule compounds, compounds from a compound library, etc.

[0023] In another aspect of the invention, the use of the GA2ox9 gene of grasses or the protein encoded therein is provided for use as a molecular marker for identifying seed dormancy and seed germination in plants (e.g., a tracking marker for desirable traits in offspring).

[0024] In another aspect of the invention, a method is provided for targeted selection of grass plants or seeds whose seed dormancy and seed germination are regulated, the method comprising: identifying the expression or activity of the GA2ox9 gene or its encoded protein in a test plant or its seeds; if the expression or activity of the GA2ox9 gene or its encoded protein in the test plant or its seeds is higher than the average expression / activity value of such plants or seeds, then it is a plant with increased seed dormancy or reduced germination; if the expression or activity of the GA2ox9 gene or its encoded protein in the test plant or its seeds is significantly lower than the average expression / activity value of such plants or seeds, then it is a plant or its seeds with decreased seed dormancy or promoted germination.

[0025] In one or more embodiments, the method for directional selection of plants or their seeds with regulated seed dormancy and seed germination further includes: observing changes in the signaling pathway “GA2ox9-gibberellin-α-amylase-soluble sugar-abscisic acid” involved by the GA2ox9 gene or its encoded protein.

[0026] In one or more embodiments, nucleic acid sequences are identified using methods including (but not limited to): sequencing, PCR amplification, restriction enzyme digestion analysis, probe methods, hybridization, microarray methods, and allele polymorphism analysis.

[0027] In one or more embodiments, the GA2ox9 is derived from or includes (but is not limited to) the following grasses: rice (Oryza sativa), millet (Setaria italica), wheat (Triticum aestivum), maize (Zeamays), sorghum (Sorghum bicolor), barley (Hordeum vulgare), millet (Panicum miliaceum), rye (Secale cereale), oats (Avena sativa L.), and two-stalked short-stalked grass (Brachypodium distachyum).

[0028] In one or more embodiments, the protein encoded by the GA2ox9 gene is selected from: (a) a protein with the amino acid sequence shown in SEQ ID NO:2; (b) a protein derived from (a) having the function of (a) formed by substituting, deleting, or adding one or more (e.g., 1-30 or 1-20; preferably 1-10; more preferably 1-5, 1-3, or 1-2) amino acid residues of the amino acid sequence shown in SEQ ID NO:2; (c) a protein having the function of (a) having an amino acid sequence that is 80% or more (preferably 85%, 90%, or 95%; e.g., 98% or 99%) identical to the amino acid sequence defined in (a); or (d) a fragment of SEQ ID NO:2 having the function of (a); or (d) a protein formed by adding a tag sequence to the N or C terminus of the protein with the amino acid sequence shown in SEQ ID NO:2, or by adding a signal peptide sequence to its N terminus.

[0029] In one or more embodiments, the GA2ox9 gene or the protein it encodes includes its homologs.

[0030] In one or more embodiments, the plant is a plant that expresses GA2ox9 or its homologs.

[0031] In one or more embodiments, the plant is a plant in which seed germination and / or dormancy are regulated by gibberellin and abscisic acid.

[0032] In one or more embodiments, high expression (or high expression) or high activity (or high activity) means a statistically significant increase in expression or activity compared to the average expression or activity of similar or identical plants, such as an increase of 10%, 20%, 40%, 60%, 80%, 90%, or higher.

[0033] In one or more embodiments, the low expression (or low expression) or low activity (or low activity) refers to a statistically significant reduction in expression or activity compared to the average expression or activity of similar or identical plants, such as a reduction of 10%, 20%, 40%, 60%, 80%, 90%, or lower.

[0034] In one or more embodiments, “increasing seed dormancy or reducing germination” means that, compared with the same type or the same plant, there is a statistically significant increase / promotion in seed dormancy (e.g., a 5%, 10%, 20%, 40%, 60%, 80%, 90% or higher proportion of seeds in a dormant state) or a statistically significant reduction in seed germination (e.g., a 10%, 20%, 40%, 60%, 80%, 90% or more proportion of seeds in a germinating state).

[0035] In one or more embodiments, “reducing plant seed dormancy or promoting germination” means a statistically significant decrease in seed dormancy (e.g., a decrease in the proportion of dormant seeds by 10%, 20%, 40%, 60%, 80%, 90%, or more) or a statistically significant increase in seed germination (e.g., an increase in the proportion of germinating seeds by 10%, 20%, 40%, 60%, 80%, 90%, or more) compared to a quantity of the same type or plant species.

[0036] Other aspects of the invention will be apparent to those skilled in the art from the disclosure herein. Attached Figure Description

[0037] Figure 1 Sequencing analysis of six homozygous OsGA2ox9 loss-of-function mutants with different edits. Green text indicates PAM (Protospacer adjacent motif) sequences, blue text indicates gRNA sequences, and red dots indicate regions with base deletions.

[0038] Figure 2The germination rate of seeds from OsGA2ox9 loss-of-function and overexpression transgenic materials is shown in the top figure. Seeds were collected 27-29 days after pollination, and the germination rate was recorded at the same time point for 5 consecutive days under a germination temperature of 28℃ and a 12-hour light / 12-hour dark condition. The materials used included Zhonghua 11, OsGA2ox9-Cas9 transgenic lines (L2, L4), and OsGA2ox9-OE transgenic lines (L1, L2, L3). Mature seeds of OsGA2ox9 loss-of-function transgenic materials showed germination of panicles (bottom figure).

[0039] Figure 3 The activity of α-amylase was increased in mature seeds of the OsGA2ox9-Cas9 transgenic material.

[0040] After transecting OsGA2ox9-Cas9 transgenic material and wild-type mature seeds, the endosperm portion without embryos was placed under different GA3 concentrations (0, 0.01mM, 0.1mM) to detect α-amylase activity.

[0041] Figure 4 The soluble sugar content increased in mature seeds of the OsGA2ox9-Cas9 transgenic material.

[0042] The contents of trehalose, glucose, fructose and sucrose in mature seeds of OsGA2ox9-Cas9 transgenic material were determined by HPLC.

[0043] Figure 5 Seeds of the OsGA2ox9-Cas9 transgenic material showed decreased sensitivity to ABA compared to the wild type (left figure). qPCR results showed downregulated expression of OsABI3 and OsABI5 in seeds of the OsGA2ox9-Cas9 transgenic material 18 and 28 days after pollination.

[0044] Figure 6 Homology analysis of the OsGA2ox9 gene. Detailed Implementation

[0045] Through extensive research, the inventors have revealed a novel gene, GA2ox9, that regulates seed dormancy and germination in plants. GA2ox9 regulates seed dormancy and spikelet germination by modulating gibberellin (GA) activity in the endosperm, affecting α-amylase activity and soluble sugar content, and altering abscisic acid (ABA) signaling in the embryo. This invention further reveals that deletion of the GA2ox9 gene leads to decreased seed dormancy and spikelet germination; while overexpression of the GA2ox9 gene can improve seed dormancy. This invention provides a new approach for improving seed dormancy and reducing spikelet germination in plants.

[0046] the term

[0047] As used herein, "plant" includes plants that express GA2ox9 (including its homologs) or contain GA2ox9 and the signaling pathways it participates in. According to knowledge in the art, plants expressing GA2ox9 inherently possess the mechanisms of action claimed in this invention and can achieve the technical effects claimed in this invention. In some embodiments, the plant is a crop, preferably a cereal crop, which is a crop with grains (ears). The "cereal crop" can be a grass (Poaceae). In some preferred embodiments, the grass includes: rice, barley, wheat, oats, rye, corn, sorghum, and *Brachys pubescens*. The GA2ox9 includes its homologs (homologous genes and their encoded proteins).

[0048] In some embodiments, the term "plant" refers to a plant possessing an embryo and endosperm structure. It is well known to those skilled in the art that plant embryos and endosperm have similar compositions, and plants possessing embryo or endosperm structures share common characteristics, including conserved genes or regulatory elements in their genomes that regulate gene transcription and expression, such as a series of elements that regulate the formation of the embryo or endosperm. In this invention, the term "plant" expresses or contains GA2ox9, and the signaling pathway it participates in ("GA2ox9-gibberellin-α-amylase-soluble sugar-abscisic acid") is also disclosed. Based on knowledge in the art, plants expressing GA2ox9 inherently possess the mechanism of action claimed in this invention, and can achieve the technical effects claimed in this invention.

[0049] As used herein, the terms “enhance,” “improve,” “promote,” or “enhance” are interchangeable and, in their application, should mean an increase of at least 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%, preferably at least 15% or 20%, more preferably 25% or 30%, compared to the control plant as defined herein.

[0050] Regarding "control plants," selecting appropriate control plants is a routine part of experimental design. These can include corresponding wild-type plants or transgenic plants without the target gene. Control plants are generally the same plant species or even varieties of the same species or class as the plant being evaluated. Control plants can also be individuals from transgenic plants that have lost their transgenic components due to segregation. As used in this article, control plants refer not only to whole plants but also to plant parts, including seeds and seed portions.

[0051] As used in this invention, "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.

[0052] As used in this invention, the terms "upregulation," "promotion," "enhancement," or "boost" indicate a significant upregulation, promotion, enhancement, or boost, such as an upregulation, promotion, enhancement, or boost of 20%, 40%, 60%, 80%, 90%, or higher.

[0053] As used in this invention, the terms "down-adjustment," "reduction," "suppression," "weakening," or "attenuation" refer to a significant down-adjustment, reduction, suppression, weakening, or attenuation, such as a down-adjustment, reduction, suppression, weakening, or attenuation of 20%, 40%, 60%, 80%, 90%, or lower.

[0054] GA2ox9 gene and the protein it encodes

[0055] In this invention, unless otherwise specified, the GA2ox9 protein includes its homologs (homologous proteins). The GA2ox9 can be a polypeptide (protein) having the amino acid sequence shown in SEQ ID NO:2, or it can include sequence variations having the same function as the GA2ox9 protein.

[0056] The variations include (but are not limited to): deletions, insertions, and / or substitutions of several amino acids (typically 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10, and even more preferably 1-8 or 1-5); and additions or deletions of one or more amino acids (typically up to 20, preferably up to 10, and more preferably up to 5) at the C-terminus and / or N-terminus. Any protein with high homology to the GA2ox9 protein (e.g., 70% or higher homology to the polypeptide sequence shown in SEQ ID NO:2; preferably 80% or higher; more preferably 90% or higher, such as 95%, 98%, or 99% homology) and having the same function as the GA2ox9 protein is also included in this invention.

[0057] In this invention, the term "GA2ox9 protein" also includes its homologs. It should be understood that while this invention preferably studies GA2ox9 proteins derived from specific species, other polypeptides or genes derived from other species, particularly grasses, that are highly homologous to the GA2ox9 protein (e.g., having more than 70%, more particularly 80%, 85%, 90%, 95%, or even more than 98% sequence identity) are also within the scope of this invention.

[0058] In this invention, polypeptides derived from species other than rice that have high homology with the sequence of SEQ ID NO:2 or that play the same or similar role in the same or similar signaling pathways are also included.

[0059] The present invention also provides isolated proteins, which are fragments of GA2ox9 protein or formed by adding other proteins or tags at both ends.

[0060] This invention also relates to a polynucleotide sequence encoding the GA2ox9 protein of this invention or a sequence variant thereof. The polynucleotide may be in DNA or RNA form. The DNA form includes cDNA, genomic DNA, or synthetically produced DNA. The DNA may be single-stranded or double-stranded. The DNA may be a coding strand or a non-coding strand. The coding region sequence encoding the mature polypeptide may be identical to the coding region sequence shown in SEQ ID NO:1 or a degenerate variant. As used herein, "degenerate variant" refers to a nucleic acid sequence encoding a polypeptide having the sequence of SEQ ID NO:2, but differing from the coding region sequence shown in SEQ ID NO:1. This invention also relates to variants (variants) of the aforementioned polynucleotide that encode polypeptides or fragments, analogs, and derivatives of polypeptides having the same amino acid sequence as those of this invention.

[0061] The present invention also relates to a vector containing the aforementioned polynucleotide, and a host cell genetically engineered using the aforementioned vector or polypeptide to encode nucleic acids.

[0062] In this invention, the polynucleotide sequence encoding the polypeptide of this 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 can replicate and remain stable in the host. An important characteristic of an expression vector is that it typically contains an origin of replication, a promoter, a marker gene, and translation control elements. Preferably, the expression vector may also selectively contain resistance elements, selection elements, or reporter gene elements, such as Bar or GUS.

[0063] When the aforementioned polynucleotide is expressed in higher eukaryotic cells, the insertion of an enhancer sequence into the vector will enhance transcription. An enhancer is a cis-acting factor of DNA, typically consisting of approximately 10 to 300 base pairs, that acts on the promoter to enhance gene transcription.

[0064] Transforming host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. Plant transformation can be performed using methods such as Agrobacterium-mediated transformation or gene gun transformation, including spraying, leaf disc transformation, and rice embryo transformation.

[0065] Plant Transformation

[0066] This invention, through extensive systematic research and large-scale screening, identified the GA2ox9 gene, which regulates traits such as seed dormancy and seed germination in plants. The protein encoded by the GA2ox9 gene increases seed dormancy or reduces germination by decreasing the content of active gibberellins (preferably gibberellins in the seed endosperm), reducing α-amylase activity, decreasing soluble sugar content, and increasing abscisic acid (preferably abscisic acid in the embryo) sensitivity. Conversely, downregulating the GA2ox9 gene increases the content of active gibberellins (preferably gibberellins in the seed endosperm), increases α-amylase activity, increases soluble sugar content, decreases abscisic acid (preferably abscisic acid in the embryo) sensitivity, and reduces seed dormancy or promotes germination.

[0067] Seed dormancy and germination are regulated by complex mechanisms involving diverse pathways and genes / proteins. Abscisic acid (ABA) and gibberellin (GA) are two plant hormones that regulate seed dormancy and germination through antagonism. ABA plays two main roles in seed dormancy: inhibiting seed germination and inducing dormancy during seed maturation. GA also plays two main roles: promoting embryo growth potential and removing the mechanical barrier formed by the endosperm during germination by increasing α-amylase activity. ABA is an inducing factor for seed dormancy. In plant seeds, ABA synthesis involves multiple enzymes, and changes in their activity lead to changes in ABA content in the seed, thereby regulating seed dormancy. Besides ABA metabolic pathways, ABA signaling induces the expression of downstream genes through the PYR / PYL / RCAR-PP2C-SnRKs cascade, thus affecting seed dormancy. GA promotes radicle growth, seed coat penetration, and elongation during seed germination. Seeds respond to GA signals by activating the expression of germination-related genes, inducing the production of cell wall remodeling enzymes, and weakening the endosperm and cortex surrounding the embryo, which facilitates the emergence of the radicle from the seed coat. Both GA metabolism and GA signal transduction processes are involved in the regulation of seed dormancy.

[0068] This invention proposes for the first time that the GA2ox9 gene has a synergistic effect with GA and ABA, thereby proposing a novel signaling pathway: the GA2ox9-gibberellin-α-amylase-soluble sugar-abscisic acid pathway.

[0069] Based on the inventor's new discovery, a use of GA2ox9 or its regulators is provided for regulating seed dormancy and seed germination in grasses. The GA2ox9 includes its homologs.

[0070] It should be understood that, after learning about the regulatory role of GA2ox9 in grasses, various methods well known to those skilled in the art can be used to regulate the expression or activity of GA2ox9 as needed, and these methods are all included in this invention.

[0071] GA2ox9 activity can be upregulated using upregulators of its expression or activity. These upregulators include promoters, agonists, and activators. The terms "upregulation" and "promotion" refer to either the upregulation or promotion of protein activity or protein expression. Any substance that can increase the activity of the GA2ox9 protein, improve the stability of the GA2ox9 gene or protein, upregulate the expression of the GA2ox9 gene, or increase the effective duration of action of the GA2ox9 protein can be used in this invention as a useful substance for upregulating the GA2ox9 gene or its encoded protein. These substances can be compounds, small chemical molecules, or biomolecules. The biomolecules can be at the nucleic acid level (including DNA and RNA) or at the protein level.

[0072] As a preferred embodiment, a method for upregulating the expression or activity of GA2ox9 in plants is provided, the method comprising: transferring an expression construct or vector containing the GA2ox9 gene into plants.

[0073] Preferably, a method for preparing transgenic plants is provided, comprising:

[0074] (1) Transferring exogenous nucleic acid encoding GA2ox9 into plant organs or tissues to obtain plant tissues or organs transformed with said nucleic acid; and

[0075] (2) The plant tissues or organs that have been transferred with exogenous nucleic acids obtained in step (1) are regenerated into plant plants.

[0076] As a preferred example, the method includes the steps of:

[0077] (s1) Provide Agrobacterium carrying an expression vector, said expression vector containing exogenous (recombinant) GA2ox9;

[0078] (s2) Contact the plant tissue or organ with the Agrobacterium in step (s1) so that the GA2ox9 is transferred into and integrated into the chromosome of the plant cell;

[0079] (s3) Select plant cells, tissues or organs that have been transferred into the GA2ox9.

[0080] The present invention also includes plants obtained using any of the foregoing methods, said plants including: transgenic plants introduced with said GA2ox9.

[0081] In this invention, the downregulator of GA2ox9 protein or its encoding gene refers to any substance that can reduce the activity of GA2ox9 protein, reduce the stability of GA2ox9 protein or its encoding gene, downregulate the expression of GA2ox9 protein, reduce the effective action time of GA2ox9 protein, inhibit the transcription and translation of GA2ox9 gene, or reduce the phosphorylation / activation level of the protein. These substances can all be used in this invention as substances useful for downregulating GA2ox9 protein. They can be compounds, small chemical molecules, or biomolecules. The biomolecules can be at the nucleic acid level (including DNA and RNA) or at the protein level. For example, the downregulator is: interfering RNA molecules or antisense nucleotides that specifically interfere with the expression of GA2ox9 protein or other signaling pathway genes; or gene editing reagents that specifically edit the GA2ox9 gene, etc.

[0082] As a preferred embodiment of the present invention, a method for downregulating GA2ox9 protein in plants is provided, comprising targeted mutation, gene editing, or gene recombination of the GA2ox9 protein to achieve downregulation. As a more specific embodiment, any of the above methods is used to transform the GA2ox9 protein into its mutant form, thereby rendering it ineffective. As a more specific embodiment, gene editing is performed using a CRISPR / Cas9 system. Suitable sgRNA target sites result in higher gene editing efficiency; therefore, suitable target sites can be designed and identified before gene editing. After designing specific target sites, in vitro cell activity screening is required to obtain effective target sites for subsequent experiments. Preferred gene editing reagents are provided in the embodiments of the present invention.

[0083] As an alternative approach, the method for downregulating the expression of GA2ox9 protein in plants may include: (1) transferring an interfering molecule that interferes with the expression of the GA2ox9 gene into plant cells, tissues, organs, or seeds to obtain plant cells, tissues, organs, or seeds transformed with the interfering molecule; (2) regenerating plants from the plant cells, tissues, organs, or seeds transformed with the interfering molecule obtained in step (1). Preferably, the method further includes: (3) selecting plant cells, tissues, or organs transformed with the vector.

[0084] The method can be implemented using any appropriate conventional means, including reagents, temperature, pressure conditions, etc.

[0085] Plant targeted screening and molecular markers

[0086] Based on the inventors' new findings, this invention provides a molecular marker suitable for identifying plant traits, namely the GA2ox9 gene; these plant traits include seed dormancy and seed germination. This invention also relates to specific molecular markers designed for the GA2ox9 gene, and identification strategies.

[0087] As a preferred embodiment, the method of the present invention for targeted selection or identification of plants with regulated agronomic traits includes: identifying the expression or activity of the GA2ox9 gene or its encoded protein in the test plant or its seeds; if the expression or activity of the GA2ox9 gene or its encoded protein in the test plant or its seeds is higher than the average expression / activity value of such plants or their seeds, then it is a plant with increased seed dormancy or reduced germination; if the expression or activity of the GA2ox9 gene or its encoded protein in the test plant or its seeds is significantly lower than the average expression / activity value of such plants or their seeds, then it is a plant or its seeds with reduced seed dormancy or promoted germination.

[0088] Based on the novel findings of this invention, those skilled in the art can employ any of the various techniques known in the art or under development to analyze nucleic acid sequences, and these techniques are all included in this invention. The methods described include, but are not limited to: sequencing, PCR amplification, probe methods, hybridization, restriction enzyme digestion analysis, allele polymorphism analysis (such as melting curve analysis) for nucleic acid sequence identification, etc.

[0089] This invention has promising applications in molecular design breeding and crop variety improvement using genetic engineering technology.

[0090] After understanding the function of the GA2ox9 gene, it can be used as a molecular marker for targeted plant screening. This new discovery can also be used to screen for substances or potential substances that can regulate seed dormancy and germination in grasses by modulating this mechanism.

[0091] This invention provides a method for screening substances (potential substances) that regulate traits of grass plants, comprising: (1) adding a candidate substance to a system containing the GA2ox9 gene or its encoded protein; (2) detecting the system and observing the expression or activity of the GA2ox9 gene or its encoded protein; if the candidate substance upregulates the GA2ox9 gene or its encoded protein, it indicates that the candidate substance is a regulator that increases seed dormancy or reduces germination; if the candidate substance downregulates the expression or activity of the GA2ox9 gene or its encoded protein, it indicates that the candidate substance is a regulator that reduces plant seed dormancy or promotes germination.

[0092] Methods for screening substances that act on proteins or genes or specific regions thereof as targets are well known to those skilled in the art, and these methods can all be used in this invention. The candidate substances can be selected from: peptides, polymeric peptides, peptide-like substances, non-peptide compounds, carbohydrates, lipids, antibodies or antibody fragments, ligands, small organic molecules, small inorganic molecules, and nucleic acid sequences, etc. Depending on the type of substance to be screened, those skilled in the art understand how to select an appropriate screening method.

[0093] The detection of protein-protein interactions and their strength can be achieved using a variety of techniques well-known to those skilled in the art, such as GST-Pull Down, bimolecular fluorescence complementation assays, yeast two-hybrid systems, or immunoprecipitation techniques.

[0094] Through large-scale screening, a class of substances that specifically act on the GA2ox9 protein or its encoding gene and have a regulatory effect on the improvement of traits in grass plants can be obtained.

[0095] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed according to conventional conditions such as those described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd Edition, Science Press, or according to the manufacturer's recommendations.

[0096] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0097] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0098] Sequence information

[0099] OsGA2ox9 gene CDS sequence (SEQ ID NO:1):

[0100]

[0101] OsGA2ox9 protein sequence (SEQ ID NO:2):

[0102] ATTERYSVAYFLCPSYDSPIGTCREPSPYKAFTFGEYRRRVQEDVKKTGKKTGLSNFLV*

[0103] Example 1: Changes in dormancy and germination of seeds from transgenic materials with OsGA2ox9 gene deletion and overexpression.

[0104] 1. Preparation of transgenic material with OsGA2ox9 gene deletion

[0105] Gene editing technology is used to specifically edit OsGA2ox9 in the genome. A single guide RNA (sgRNA) that specifically recognizes OsGA2ox9 exons is selected, which carries the Cas9 protein to the target location in the OsGA2ox9 genome. The Cas9 protein cuts the double-stranded DNA and creates a gap. The plant itself repairs the gap by joining non-homologous ends, ultimately resulting in the insertion or deletion of bases.

[0106] The sequence of the sgRNA is: cctcaacgactcctaccgct (SEQ ID NO:3).

[0107] To generate sgRNA, two knockout primers were designed, phosphorylated, annealed, and paired, and then ligated to pOs-sgRNA. The pOs-sgRNA and pUbi-Cas9 were then mixed and transferred to the pUbi-Cas9 vector using the LR reaction. The vector was identified by colony PCR and sequencing, and finally the GA2ox9-CRISPR-Cas9 binary vector was obtained.

[0108] The GA2ox9-CRISPR-Cas9 binary vector was introduced into Agrobacterium tumefaciens EHA105 (purchased from Ingenium Biotech, USA), transformed into rice callus tissue, and positive transgenic lines were obtained through resistance selection. The T0 and T1 generations of transgenic plants were sequenced to obtain six homozygous transgenic materials with different OsGA2ox9 deletion functions, such as... Figure 1 The plants were named OsGA2ox9-Cas9-1~6 (plants L1~L6). The inventors selected L2 and L4 for subsequent experiments.

[0109] 2. Preparation of transgenic materials overexpressing the OsGA2ox9 gene

[0110] The upstream promoter sequence of OsGA2ox9 (1351 bp before the ATG start codon) and the CDS sequence of OsGA2ox9 (1077 bp) were amplified by PCR. The promoter and CDS of the OsGA2ox9 gene were ligated to the backbone fragment of the pCAMBIA1300 vector using T4 ligase. This ligation was performed on *E. coli* DH5α, and the plasmid was further extracted, digested with enzymes for verification, and sequenced. The recombinant plasmid pGA2ox9-GA2ox9-pCAMBIA1300, which contained the OsGA2ox9 gene and was confirmed by sequencing, was introduced into *Agrobacterium* EHA105 and transformed into rice callus tissue. Positive transgenic lines were obtained through resistance selection. After T1 and T2 generations, eight independent homozygous OsGA2ox9 overexpression (OE) transgenic lines were obtained using resistance and PCR identification, named OsGA2ox9-OE-1 to OsGA2ox9-8.

[0111] 3. Phenotypic analysis of transgenic materials with OsGA2ox9 gene deletion and overexpression

[0112] Homozygous transgenic materials of OsGA2ox9-Cas9 and OsGA2ox9-OE were planted in the experimental field and harvested after normal growth and fruiting. At the same time, wild-type Zhonghua 11 was planted as a control.

[0113] Collect seeds 27-29 days after pollination, placing 50 seeds per group in a round dish, with 3 replicates per material. Soak the seeds in sterile water, and place the round dishes in a 28℃, 12-hour light / 12-hour dark incubator for 5 days, changing the water daily and counting the number of germinating seeds.

[0114] The results showed that the OsGA2ox9-Cas9 transgenic material had a faster germination rate than the wild type, reaching 80% germination rate after 4 days of water absorption, and mature seeds exhibited ear germination; while the OsGA2ox9 overexpression material had a slower germination rate, with a germination rate of less than 40% after 4 days of water absorption. Figure 2 .

[0115] The above results indicate that OsGA2ox9 plays an important role in seed dormancy and germination.

[0116] Therefore, OsGA2ox9 helps to keep plant seeds in a dormant state and reduces spikelet germination, which is beneficial when long-term seed preservation is required. Conversely, downregulating OsGA2ox9 in plants can promote seed germination and spikelet germination.

[0117] Example 2: Changes in the content of active gibberellin (GA) in the OsGA2ox9 transgenic material, and changes in the α-amylase activity and soluble sugar content in the seeds.

[0118] 1. OsGA2ox9 functions as a GA2 oxidase.

[0119] The inventors selected rice seedlings with high levels of active GA to determine the function of the protein encoded by the OsGA2ox9 gene and to observe whether it is a GA2 oxidase (which can convert active GA precursors into an inactive state).

[0120] Seedlings of OsGA2ox9-Cas9 and OsGA2ox9-OE that have grown for 14 days were used to determine the GA content using high performance liquid chromatography.

[0121] The results showed that the OsGA2ox9-Cas9 transgenic line had an increased content of active GAs:GA4 and a decreased content of inactive GAs:GA34 and GA51 compared to the wild type; while the OsGA2ox9-OE transgenic line had a decreased content of active GAs:GA1 and GA3 and an increased content of inactive GAs:GA51 compared to the wild type.

[0122] These results indicate that OsGA2ox9 functions as a GA2 oxidase, altering the levels of active and inactive GA: downregulation of OsGA2ox9 increases the content of active GA in plants, while upregulation of OsGA2ox9 decreases the content of active GA in plants.

[0123] 2. Amylase activity

[0124] Active GA can enhance α-amylase activity. Based on the principle that starch turns blue when it comes into contact with iodine, adding an appropriate amount of starch to the culture medium, if the α-amylase activity in the seeds is strong, will catalyze the decomposition of starch, leading to the appearance of "white spots". The inventors placed the embryo-removed seeds of wild-type and OsGA2ox9-Cas9 transgenic materials on agar medium containing 0.2% soluble starch and treated them at 30°C in the dark for 48 hours. After removing the seeds, the culture medium was stained with iodine / potassium iodide solution.

[0125] The results showed that the OsGA2ox9-Cas9 transgenic material seeds exhibited a more pronounced "white spot" phenomenon, indicating that the α-amylase activity in the seeds of the OsGA2ox9-Cas9 transgenic material was higher than that of the wild type. Figure 3 .

[0126] GA induces the expression of α-amylase, which can cleave the α-1,4-glucan bonds in starch, hydrolyzing starch into soluble sugars. The inventors used liquid chromatography to detect the content of four soluble sugars (trehalose, glucose, fructose, and sucrose) in mature seeds of the OsGA2ox9-Cas9 transgenic material.

[0127] The results showed that the mature seeds of the OsGA2ox9-Cas9 transgenic material contained increased levels of glucose, fructose, and sucrose compared to the wild type. Figure 4 .

[0128] Example 3: Altered ABA signaling in OsGA2ox9 transgenic material affects seed dormancy and germination.

[0129] 1. Sensitivity analysis of ABA

[0130] The inventors treated wild-type and OsGA2ox9-Cas9 transgenic seeds 27-29 days after pollination with different concentrations of ABA (0, 5 μM, 10 μM), and the germination rate was measured under the same conditions as in Example 1.

[0131] The results showed that the OsGA2ox9-Cas9 transgenic seeds were less sensitive to ABA than the wild type.

[0132] 2. qPCR analysis of key factors in the ABA signaling pathway

[0133] Increased soluble sugar content in seeds inhibits the expression of key ABA signaling pathway factors ABI3 and ABI5. Therefore, the inventors analyzed the expression of key ABA signaling pathway factors in the OsGA2ox9-Cas9 transgenic material using qPCR.

[0134] qPCR showed that the expression of OsABI3 and OsABI5 was downregulated in the seeds of the OsGA2ox9-Cas9 transgenic material, such as... Figure 5 .

[0135] This indicates that OsGA2ox affects α-amylase activity and soluble sugar content by regulating the content of active GA, thereby altering ABA signaling in the embryo (which is sensitive to ABA) and seed dormancy.

[0136] Example 4: Conservation analysis of OsGA2ox9 in different species

[0137] The inventors analyzed the conservation of OsGA2ox9 in different species. The results showed that homologous genes exist in various plants such as Arabidopsis thaliana, wheat, and maize, for example... Figure 6 .

[0138] Sprouting at the spike not only affects yield but also severely reduces seed quality. Varieties with strong dormancy are resistant to sprouting at the spike. Isolating key regulatory genes for seed dormancy is crucial for discovering gene resources that resist sprouting at the spike. Homology analysis shows that OsGA2ox9 has homologous genes in various plants, including Arabidopsis thaliana, wheat, and maize.

[0139] The results of enhanced seed dormancy and delayed germination in transgenic materials overexpressing OsGA2ox9 suggest that OsGA2ox9 or its homologs can be used to improve seed dormancy (putting seeds into a dormant state) and reduce ear germination.

[0140] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims. Furthermore, all documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference.

Claims

1. A method for regulating seed dormancy and seed germination in grasses, comprising: Regulating plants GA2ox9 The expression or activity of a gene or its encoded protein regulates seed dormancy and seed germination in plants; the grass shown is rice; the method is as follows: (a) Overexpression GA2ox9 Genes that increase seed dormancy or reduce germination; and (b) Knockout GA2ox9 Genes that reduce seed dormancy or promote germination in plants; The GA2ox9 The protein encoded by the gene has an amino acid sequence as shown in SEQ ID NO:

2.

2. The method as described in claim 1, characterized in that, The overexpression GA2ox9 Genes include: GA2ox9 The gene, or an expression construct or vector containing the gene, is transferred into the plant.

3. The method as described in claim 1, characterized in that, The knockout GA2ox9 Genes include: gene knockouts achieved through gene editing using the CRISPR system. GA2ox9 Gene.

4. The method as described in claim 1, characterized in that, The knockout method includes: […]. GA2ox9 Genes are modified to cause frameshifts or premature terminators.

5. The method as described in claim 4, characterized in that, The modification is targeted at positions 367-410 of the nucleotide sequence shown in SEQ ID NO:

1.

6. The method as described in claim 3, characterized in that, The nucleotide sequence of the sgRNA used in the gene editing is shown in SEQ ID NO:

3.

7. The method as described in claim 1, characterized in that, GA2ox9 Genes or their encoded proteins can reduce seed dormancy or germination by decreasing the content of active gibberellins, reducing α-amylase activity, reducing soluble sugar content, increasing abscisic acid sensitivity.

8. The method as described in claim 1, characterized in that, Lower GA2ox9 Genes or their encoded proteins can increase the content of active gibberellins, increase α-amylase activity, increase soluble sugar content, reduce abscisic acid sensitivity, reduce plant seed dormancy, or promote germination.

9. A kind GA2ox9 The purpose of this gene is to regulate seed dormancy and germination in plants; the plant shown is rice; among which: (a) Overexpression GA2ox9 Genes that can increase seed dormancy or reduce germination; and (b) Knockout GA2ox9 Genes that reduce seed dormancy or promote germination in plants; The GA2ox9 The protein encoded by the gene has an amino acid sequence as shown in SEQ ID NO: 2.

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