Gene for regulating seed dormancy and germination of gramineae plant and use thereof

Abstract

Provided are a gene for regulating seed dormancy and germination of a gramineae plant and the use thereof. Seed dormancy and seed germination in plants are regulated by means of regulating the expression or activity of a plant ERF04 gene or a protein encoded thereby. The deletion of the ERF04 gene leads to an increase in seed dormancy and reduces the pre-harvest sprouting phenomenon. By means of the provided method, a plant strain with enhanced dormancy can be obtained, and the pre-harvest sprouting resistance thereof can be effectively improved, providing a new approach for improving the seed dormancy and reducing the pre-harvest sprouting in plants.

Description

Gene for regulating seed dormancy and germination of gramineae and application thereof TECHNICAL FIELD

[0001] The present application relates to the field of botany and genetic engineering, in particular to a gene for regulating seed dormancy and germination of gramineae and application thereof. BACKGROUND

[0002] Seed dormancy refers to the phenomenon that seeds with germination ability cannot initiate germination even in the case that the external environmental conditions are suitable, and it is the inherent property of most higher plant seeds. Until the dormant seeds are broken, the germination process can be initiated. Seed germination refers to the process that dry and static seeds are fully absorbed after being soaked, and finally the radicle or plumule breaks through the seed coat, fruit coat and even the maternal tissue such as glume. In the process of crop domestication and breeding, varieties with fast and uniform germination are more favored, so the moderate dormancy of seeds is often ignored, resulting in that the main varieties of many crops such as rice and wheat are prone to serious pre-harvest sprouting problem in production.

[0003] Pre-harvest sprouting (PHS) refers to the phenomenon that seeds on the ear germinate before harvesting due to high temperature and high humidity environmental conditions. The phenomenon of pre-harvest sprouting has been concerned as early as the 1980s of last century, and multiple QTLs related to pre-harvest sprouting have been located, which provides clues for subsequent key gene mining and cloning. When pre-harvest sprouting occurs, the starch stored in the endosperm of crop seeds begins to degrade, so once pre-harvest sprouting occurs, it will cause loss to crop production, reduce yield, grain quality and seed value. The analysis of seed dormancy and germination mechanism can provide theoretical guidance and genetic resources for breeding pre-harvest sprouting resistant crop varieties. The regulation of pre-harvest sprouting is a complex physiological process, and there are many influencing factors. In addition to external factors such as water, temperature, light, etc., it is also affected by hormones, sugars, active oxygen, NO, microRNA, etc. in the seed.

[0004] The gradual solution of the problem of grain pre-harvest sprouting must rely on the analysis of the molecular mechanism of seed dormancy and germination process. The in-depth and systematic molecular level research of this process is mainly completed in the model plant Arabidopsis thaliana, and the research progress in gramineae crops such as rice and wheat is relatively slow. Most of the genes reported are mutated to show the phenotype of pre-harvest sprouting, and overexpression is needed to produce pre-harvest sprouting resistance, which is more difficult to apply to crop breeding. Therefore, mining specific inhibitors of grain dormancy is of great significance for in-depth analysis of the molecular regulation network of this biological process and breeding of pre-harvest sprouting resistant rice and wheat varieties.

[0005] SUMMARY

[0006] In view of the defects in the prior art, the present application provides a gene for regulating seed dormancy and germination of a plant in the family Poaceae and an application thereof.

[0007] The present application provides a method for regulating seed dormancy and germination of a plant in the family Poaceae, and the method comprises regulating expression or activity of a plant ERF04 gene or a protein encoded thereby, so as to regulate seed dormancy and germination of the plant.

[0008] In one or more embodiments, the method is selected from:

[0009] (i) up-regulating expression or activity of the ERF04 gene or the protein encoded thereby, so as to reduce seed dormancy or promote germination;

[0010] (ii) down-regulating expression or activity of the ERF04 gene or the protein encoded thereby, so as to increase seed dormancy or reduce germination of the plant.

[0011] In one or more embodiments, the down-regulating expression or activity of the ERF04 gene or the protein encoded thereby comprises any one of knocking out or silencing the ERF04 gene in the plant, inhibiting activity of the ERF04 protein, or editing a promoter of the ERF04 gene to reduce expression of the ERF04 gene.

[0012] In one or more embodiments, the up-regulating expression or activity of the ERF04 gene or the protein encoded thereby comprises any one of:

[0013] (1) introducing an expression construct or a vector of the ERF04 gene or a promoter of the ERF04 gene into the plant;

[0014] (2) introducing an expression construct or a vector containing the ERF04 gene or containing a promoter of the ERF04 gene into the plant.

[0015] The present application also provides a method for screening a regulator for regulating seed dormancy and germination of a plant in the family Poaceae, and the method comprises: adding a candidate substance to a system containing the ERF04 gene or the protein encoded thereby; detecting the system to observe expression or activity of the ERF04 gene or the protein encoded thereby.

[0016] If the candidate substance up-regulates the ERF04 gene or the protein encoded thereby, it indicates that the candidate substance is a regulator for reducing seed dormancy or promoting germination; if the candidate substance down-regulates expression or activity of the ERF04 gene or the protein encoded thereby, it indicates that the candidate substance is a regulator for increasing seed dormancy or reducing germination of the plant.

[0017] In one or more embodiments, the dosage form of the regulator is selected from any one or more of a solution, an emulsion, a suspension, a powder, a foam, a paste, a granule, and an aerosol.

[0018] The application also provides a selection method of a plant in the family Poaceae, comprising the following steps: regenerating any one of the following mutant plant tissues or plant cells into a plant body to obtain the selected plant in the family Poaceae:

[0019] (1) knocking down the ERF04 gene;

[0020] (2) knocking out the ERF04 gene;

[0021] (3) editing the promoter of the ERF04 gene to reduce the expression of the ERF04 gene.

[0022] The application also provides a method for producing grain, comprising the following steps: planting a crop and harvesting the grain of the crop; the expression or activity of the ERF04 gene or the protein encoded by the ERF04 gene in the crop is reduced.

[0023] The application also provides the use of any one of the ERF04 gene, the protein encoded by the ERF04 gene, the promoter of the ERF04 gene, or the modulator thereof in plant breeding.

[0024] In one or more embodiments, the modulator is the ERF04 gene or the protein encoded by the ERF04 gene, an expression construct or a vector containing the ERF04 gene or the promoter of the ERF04 gene, which is used to reduce seed dormancy or promote germination.

[0025] or the modulator is a down-regulator of the ERF04 gene or the protein encoded by the ERF04 gene, which is used to increase seed dormancy or reduce germination.

[0026] In one or more embodiments, the modulator further comprises other substances for regulating plant traits.

[0027] In one or more embodiments, the other substances for regulating plant traits include an osmotic regulator, brassinolide, alginin, a fertilizer with high potassium or nitrogen or phosphorus content, trace elements (such as boron and zinc), a triazole fungicide (such as difenoconazole, propiconazole, and tebuconazole), a high-potassium foliar fertilizer, a plant hormone (such as abscisic acid, ethylene, a cytokinin, and polyamine), a rare earth element, paclobutrazol (PP333), benzoic acid, salicylic acid, and uniconazole.

[0028] In another preferred embodiment, the osmotic regulator is selected from the following group: an inorganic regulator, an organic regulator, a growth regulator, or a combination thereof.

[0029] In one or more embodiments, the plant is any one or more of Oryza sativa, Triticum aestivum, Secale cereale, Hordeum vulgare, Zea mays, Sorghum bicolor and Avena sativa L.

[0030] In one or more embodiments, the amino acid sequence of the protein encoded by the ERF04 gene is selected from any one of:

[0031] (a) any one of SEQ ID NO. 24, SEQ ID NO. 30, SEQ ID NO. 31, SEQ ID NO. 32;

[0032] (b) a protein derived from (a) by substitution, deletion, and / or addition of one or more amino acid residues in the amino acid sequence of any one of SEQ ID NO. 24, SEQ ID NO. 30, SEQ ID NO. 31, SEQ ID NO. 32, and which has the function of the protein of (a);

[0033] (c) a protein of any one of SEQ ID NO. 24, SEQ ID NO. 30, SEQ ID NO. 31, SEQ ID NO. 32 with a tag sequence added to the N or C terminus thereof, or a protein formed by adding a signal peptide sequence to the N terminus thereof;

[0034] (d) a polypeptide having >80% (preferably >90%, more preferably >95% or >98%) homology to the amino acid sequence of any one of SEQ ID NO. 24, SEQ ID NO. 30, SEQ ID NO. 31, SEQ ID NO. 32, and which has the ERF04 activity.

[0035] In one or more embodiments, the nucleotide sequence of the ERF04 gene is selected from any one of:

[0036] (i) a polynucleotide encoding a polypeptide as set forth in SEQ ID NO. 24, SEQ ID NO. 30, SEQ ID NO. 31, or SEQ ID NO. 32;

[0037] (ii) a polynucleotide having a sequence as set forth in SEQ ID NO. 21, SEQ ID NO. 27, SEQ ID NO. 28, or SEQ ID NO. 29;

[0038] (iii) a polynucleotide having a nucleotide sequence with >75% (preferably >80%, more preferably >90% or >95%) homology to the sequence shown in SEQ ID NO. 21, SEQ ID NO. 27, SEQ ID NO. 28 or SEQ ID NO. 29;

[0039] (iii) a polynucleotide having a nucleotide sequence with >75% (preferably >80%, more preferably >90% or >95%) homology to the sequence shown in SEQ ID NO. 21, SEQ ID NO. 27, SEQ ID NO. 28 or SEQ ID NO. 29;

[0040] (iii) a polynucleotide having a nucleotide sequence with >75% (preferably >80%, more preferably >90% or >95%) homology to the sequence shown in SEQ ID NO. 21, SEQ ID NO. 27, SEQ ID NO. 28 or SEQ ID NO. 29;

[0041] In one or more embodiments, the nucleotide sequence of the ERF04 gene promoter is selected from any one of the following:

[0042] (1) the nucleotide sequence shown in SEQ ID NO. 5;

[0043] (2) a nucleotide sequence with >75% (preferably >80%, more preferably >90% or >95%) homology to the sequence shown in SEQ ID NO. 5.

[0044] In summary, compared with the prior art, the present application achieves the following technical effects:

[0045] 1. The present application utilizes the ERF04 gene and the protein encoded thereby to participate in the regulation of seed dormancy in plants of the Poaceae family, and through mutation of the nucleotide sequence of the gene, a strain with resistance to sprouting of the ear is screened, which has very important application in agricultural production.

[0046] 2. The present application provides an efficient breeding method for creating a non-traditional transgenic plant variety, germplasm resource or hybrid parent with resistance to sprouting of the ear and without exogenous fragments based on the ERF04 gene. BRIEF DESCRIPTION OF DRAWINGS

[0047] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings needed in the embodiments will be briefly introduced below, and it should be understood that the following drawings only show some embodiments of the present application, and therefore should not be considered as a limitation on the scope, and other related drawings can also be obtained by those skilled in the art without creative effort on the basis of these drawings.

[0048] Figure 1 is the expression profile of OsERF04 in different tissues and different development periods of rice according to the embodiment of the present application;

[0049] Wherein Leaf is leaf; Root is root; P, Panicle, is different length of spikelet; Anther is anther; Ovary is ovule; C2, C4, C7, C11, C14, C21 and C31, respectively represent the caryopsis of 4, 7, 11, 14, 21 and 31 days after pollination; Em, Embryo, embryo; Al, Aleurone, aleurone; SE, Starchy endosperm, starchy endosperm; DAF, Days after fertilization, days after fertilization; HAI, Hours after imbibition, hours after imbibition.

[0050] Figure 2 is the GUS staining result of the transgenic rice of P-OsERF04 in the embodiment 4 of the present application.

[0051] Wherein, A-F are respectively the longitudinal section of caryopsis of 4 days, 7 days, 11 days, 14 days, 21 days and 28 days after fertilization; G-L are respectively the transverse section of caryopsis of 4 days, 7 days, 11 days, 14 days, 21 days and 28 days after fertilization; M: the transverse section of leaf, N: the transverse section of leaf sheath, O: the transverse section of node, P: the transverse section of leaf sheath wrapped by internode and outside of inflorescence axis, Q: floret and R: stamen and pistil; DA: dorsal aleurone; SE: starchy endosperm; EM: embryo; the scale of A-L picture is 1mm, and the scale of M-R picture is 2mm.

[0052] Figure 3 is the relative expression amount of OsERF04 in wild type and pUBi-OsERF04-RNAi transgenic plants in the embodiment 5 of the present application.

[0053] Figure 4 is the germination phenotype analysis of pUBi-OsERF04-RNAi transgenic plants in the embodiment 6 of the present application; Figures D, E and F are taken at 6 days after soaking; the numerical value = mean ± standard deviation (three biological repeats, 10 grains for each repeat); the scale of all pictures is 1cm; DAI: days after soaking.

[0054] Figure 5 is the mutation site information and predicted truncated protein amino acid sequence of oserf04-1 and oserf04-2 in the embodiment 7 of the present application.

[0055] Figure 6 is the germination phenotype and statistical results of oserf04-1 and oserf04-2 mutants of the embodiment 8 of the present application; wherein, A is the phenotype of the delayed germination of the grains of Zhonghua 11 and oserf04-1 and oserf04-2 mutants 45 days after pollination; B is the result picture of the grains of Zhonghua 11 and oserf04-1 and oserf04-2 mutants after 6 days of soaking; C is the statistical analysis of the germination rate of the ears of Zhonghua 11 and oserf04-1 and oserf04-2 mutants 45 days after pollination; D is the result picture of the ears of Zhonghua 11 and oserf04-1 and oserf04-2 mutants after 7 days of soaking; Bar = 0.5 cm.

[0056] Figure 7 is the result statistical of the influence of oserf04-1 and oserf04-2 mutants on other agronomic traits of the embodiment 8 of the present application; A is the picture of the plants at the grain filling stage of the control, oserf04-1 and oserf04-2 mutants; B is the statistical analysis of the plant height (B), the number of tillers (C), the ear length (D), the number of grains per ear (E), the seed setting rate (F) and the weight per 100 grains (G) of the control, oserf04-1 and oserf04-2 mutants.

[0057] Figure 8 is the relative expression amount of TaERF04-1A, TaERF04-1B and TaERF04-1D in the aleurone layer and embryo of wheat pericarp of the embodiment 10 of the present application; wherein, root is the root of 3 days after germination; shoot is the aboveground part of the seedling of 3 days after germination; stem is the stem at the grain filling stage; FL, flag leaf, flag leaf; anther is anther; pistil is pistil; S-1-2cm is 1-2 cm spike; C2, C4 and C7 are pericarp 2, 4 and 7 days after pollination; seed is the seed coat, embryo and endosperm part after removing the green pericarp; P, pericarp, pericarp; Al, aleurone, aleurone layer; MC, mature caryopses, mature pericarp. DAF, days after fertilization, days after pollination; HAI, hours after imbibition, hours after soaking.

[0058] Figure 9 is the CRISPR / Cas9 target linker primer and vector structure information of the embodiment 11 of the present application;

[0059] Figure 10 is the genotype identification result picture of TaERF04-1A, TaERF04-1B and TaERF04-1D of the embodiment 11 of the present application;

[0060] Figure 11 is the information of mutation sites of taerf04-2, taerf04-9 and taerf04-10 and the predicted amino acid sequences of truncated proteins in the embodiment 11 of the present application;

[0061] Figure 12 is the germination phenotype and statistical results of taerf04-2, taerf04-9 and taerf04-10 mutants in the embodiment 12 of the present application;

[0062] Figure 13 is the statistical results of the effects of taerf04-2, taerf04-9 and taerf04-10 mutants on other agronomic traits in the embodiment 12 of the present application. DETAILED DESCRIPTION

[0063] In order to enable persons skilled in the art to better understand the technical scheme of the present application, the technical scheme in the embodiments of the present application will be described clearly and completely below in conjunction with the accompanying drawings in the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by persons skilled in the art without creative labor should fall within the scope of protection of the present application.

[0064] TERMS

[0065] In some embodiments, the "plant" is a plant having an embryo or endosperm structure. It is well known to those skilled in the art that the components of a plant embryo or endosperm are similar, and plants having an embryo or endosperm structure have common characteristics, and there are 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 embryos or endosperms in plants. According to the knowledge in the art, plants expressing ERF04 have the mechanism claimed in the present application, and can achieve the technical effects claimed in the present application.

[0066] As used herein, the "plant" includes plants expressing ERF04 genes (including homologues thereof) or containing the signal pathways in which ERF04 genes are involved. According to the knowledge in the art, plants expressing ERF04 genes have the mechanism claimed in the present application, and can achieve the technical effects claimed in the present application.

[0067] In some embodiments, the plant is a crop, preferably a cereal crop, and the cereal crop is a crop having grains (spikelets). The "cereal crop" can be a plant in the family Poaceae. In some preferred embodiments, the plant in the family Poaceae includes rice, wheat, rye, barley, maize, sorghum and oat, etc. The ERF04 gene includes homologues thereof (homologous genes and the proteins encoded thereby).

[0068] As used herein, the terms "increasing", "improving", "promoting" or "enhancing" are interchangeable with each other and shall mean, in the application context, at least 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10% compared to a control plant as defined herein; preferably, at least 15% or 20%; more preferably, 25%, 30% or more.

[0069] With respect to "control plants", the selection of a suitable control plant is a routine part of the experimental design and can include a corresponding wild type plant or a corresponding transgenic plant without the gene of interest. The control plant is generally of the same plant species or even of the same variety as the plant to be evaluated. The control plant can also be an individual that has been isolated from the transgenic plant. The control plant as used herein refers not only to the whole plant but also to plant parts, including seeds and seed parts.

[0070] As used herein, the "grains" refer to the fruits or seeds of a plant, also known as spike grains in rice, corn, wheat, barley and the like.

[0071] As used herein, the "up-regulation", "promotion", "increase" or "enhancement" means a significant up-regulation, promotion, increase or enhancement, such as 5%, 10%, 20%, 40%, 60%, 80%, 90% or more.

[0072] As used herein, the "down-regulation", "decrease" or "inhibition" means a significant down-regulation, decrease or inhibition, such as 5%, 10%, 20%, 40%, 60%, 80%, 90% or less.

[0073] ERF04 gene and the protein encoded thereby

[0074] As used herein, the term "ERF04 gene" refers to an ERF04 gene derived from a plant (such as rice, wheat) or a variant thereof.

[0075] In a preferred embodiment, the nucleotide sequence of the ERF04 gene of the present application is any one of SEQ ID NO. 21, SEQ ID NO. 27, SEQ ID NO. 28 or SEQ ID NO. 29. Variants of the gene can be obtained by inserting or deleting regulatory regions, random or site-directed mutagenesis and the like.

[0076] The present application also includes nucleotide sequences having 80% or more homology to the preferred gene sequences of the present application (any one of SEQ ID NO. 21, SEQ ID NO. 27, SEQ ID NO. 28 or SEQ ID NO. 29) which are also effective in modulating traits in plants (e.g. rice). "Homology" refers to the level of similarity (i.e. sequence similarity or identity) between two or more nucleic acids as a percentage of positions that are the same.

[0077] It should be understood that although the genes provided in the examples of the present application are derived from rice and wheat, genes derived from other similar plants (especially plants belonging to the same family or genus as rice and wheat or other families or genera having a high degree of homology to Arabidopsis) having a certain degree of homology (conservation, such as having 80% or more, such as 85%, 90%, 95% or even 98% sequence identity) to the sequences of the present application (preferably, sequences such as any one of SEQ ID NO. 21, SEQ ID NO. 27, SEQ ID NO. 28 or SEQ ID NO. 29) are also included within the scope of the present application, provided that the skilled person can readily isolate the sequences from other plants based on the information provided in the present application after reading the present application, and methods and tools for comparing sequence identity are well known in the art, such as BLAST.

[0078] The "polynucleotide" of the present application can be in the form of DNA or RNA. The DNA form includes DNA, genomic DNA or artificially synthesized DNA, which can be single-stranded or double-stranded. The DNA can be a coding strand or a non-coding strand. The coding region sequence encoding the mature polypeptide can be identical to the coding sequence shown in SEQ ID NO. 24, SEQ ID NO. 30, SEQ ID NO. 31 or SEQ ID NO. 32 or a degenerate variant thereof.

[0079] The full-length nucleotide sequence of the present application or a fragment thereof can be obtained by PCR amplification, recombination or artificial synthesis. For PCR amplification, primers can be designed according to the nucleotide sequences disclosed in the present application, especially the open reading frame sequences, and a commercially available DNA library or a cDNA library prepared according to conventional methods known to those skilled in the art can be used as a template for amplification. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then the fragments amplified in each amplification are spliced together in the correct order. Once the relevant sequence is obtained, recombination can be used to obtain the relevant sequence in large quantities. Typically, it is cloned into a vector, then transformed into cells, and then the relevant sequence is isolated from the proliferated host cells by conventional methods.

[0080] Furthermore, the relevant sequences can be synthesized using artificial synthesis methods, especially when the length of the fragments is short. Generally, fragments of long sequences can be obtained by first synthesizing a plurality of small fragments and then connecting them. At present, the DNA sequence encoding the protein (or a fragment thereof, or a derivative thereof) of the present application can be obtained entirely by chemical synthesis. The DNA sequence can then be introduced into various existing DNA molecules (or carriers) and cells known in the art. In addition, mutations can be introduced into the protein sequence of the present application by chemical synthesis.

[0081] As used herein, the terms "polypeptide", "polypeptide having ERF04 activity", "encoded protein of ERF04 gene" are used interchangeably and refer to a polypeptide derived from ERF04 of a plant and variants thereof.

[0082] In a preferred embodiment, a typical amino acid sequence of the polypeptide of the present application is shown in any one of SEQ ID NO. 24, SEQ ID NO. 30, SEQ ID NO. 31 or SEQ ID NO. 32.

[0083] The present application relates to a polypeptide of ERF04 and variants thereof for regulating seed germination of a plant. In a preferred embodiment of the present application, the amino acid sequence of the polypeptide is shown in any one of SEQ ID NO. 24, SEQ ID NO. 30, SEQ ID NO. 31 or SEQ ID NO. 32. The polypeptide of the present application can effectively regulate seed germination of a plant.

[0084] The present application also includes polypeptides or proteins having the same or similar functions having 50% or more (preferably 60% or more, 70% or more, 80% or more, more preferably 90% or more, more preferably 95% or more, most preferably 98% or more, such as 99%) homology to the sequence shown in any one of SEQ ID NO. 24, SEQ ID NO. 30, SEQ ID NO. 31 or SEQ ID NO. 32 of the present application.

[0085] The polypeptide of the present application can be a recombinant polypeptide, a natural polypeptide, or a synthetic polypeptide. The polypeptide of the present application can be a naturally purified product, or a chemically synthesized product, or produced using a recombinant technology from a prokaryotic or eukaryotic host (e.g., bacterial, yeast, higher plant, insect, and mammalian cells). Depending on the host used in the recombinant production scheme, the polypeptide of the present application can be glycosylated, or can be non-glycosylated. The polypeptide of the present application can or can not include an initial methionine residue.

[0086] The present application also encompasses fragments and analogs of the ERF04 protein that have the biological activity of the ERF04 protein. As used herein, the terms "fragment" and "analog" refer to polypeptides that substantially retain the same biological function or activity of the native ERF04 protein of the present application.

[0087] The polypeptide fragments, derivatives or analogs of the present application can be: (i) polypeptides having one or more conservative or non-conservative amino acid substitutions (preferably conservative amino acid substitutions) which can or can not be encoded by the genetic code; (ii) polypeptides having one or more substituent groups at one or more amino acid residues; (iii) polypeptides formed by fusing the mature polypeptide to another compound, such as a compound that increases the half-life of the polypeptide, for example, a polyethylene glycol; or (iv) polypeptides formed by adding additional amino acid sequences to the N- or C-terminus of the polypeptide (such as a leader or secretory sequence, a sequence for purification of the polypeptide or a proteinaceous pro-sequence, or a fusion protein). Such fragments, derivatives and analogs are within the scope of those skilled in the art from the definition herein.

[0088] In the present application, the polypeptide variants are derived sequences of the amino acid sequence set forth in SEQ ID NO. 24, SEQ ID NO. 30, SEQ ID NO. 31 or SEQ ID NO. 32 by one or more (typically 1 to 60, preferably 1 to 30, more preferably 1 to 20, most preferably 1 to 10) substitutions, additions or deletions of at least one amino acid, and addition of one or more (typically 20 or less, preferably 10 or less, more preferably 5 or less) amino acids at the C-terminus and / or N-terminus. For example, substitution of an amino acid with a similar or similar functional amino acid, or addition of one or more amino acids at the C-terminus and / or N-terminus, typically does not change the function of the protein. These conservative variations are preferably made according to the following table:

[0089] In the present application, "promoters" are typically located near the start of transcription and are the sites to which transcription factors bind that can either promote or inhibit the process of transcription of a gene. When specific transcription factors bind to a promoter, they can either activate or repress transcription of a gene, thereby regulating protein synthesis in a cell. "ERF04 gene promoter" refers to a specific DNA region located upstream of the ERF04 gene sequence. The nucleotide sequence of the ERF04 gene promoter is selected from the group consisting of: (1) the nucleotide sequence set forth in SEQ ID NO. 5; and (2) a nucleotide sequence having >75% (preferably >80%, more preferably >90%, most preferably >95%) homology to the sequence set forth in SEQ ID NO. 5.

[0090] The present application also relates to a vector comprising the polynucleotide, and a host cell genetically engineered with the vector or the polypeptide-encoding nucleic acid.

[0091] In the present application, the polynucleotide sequence encoding the polypeptide of the present application can be inserted into a recombinant expression vector. The term "recombinant expression vector" refers to a bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus, mammalian cell virus or other vector well known in the art. In general, any plasmid and vector can be used as long as it can replicate and be stable in the host. An important feature of the expression vector is that it usually contains a replication origin, a promoter, a marker gene and a translation control element. Preferably, the expression vector can also selectively add an anti-resistance element, a screening (selection) element or a reporter gene element, such as Bar, GUS.

[0092] When the polynucleotide is expressed in higher eukaryotic cells, the transcription will be enhanced if an enhancer sequence is inserted into the vector. Enhancer is a cis-acting factor of DNA, usually about 10 to 300 base pairs, which acts on the promoter to enhance the transcription of the gene.

[0093] The transformation of host cells with recombinant DNA can be carried out by conventional techniques well known to those skilled in the art. The transformation of plants can use methods such as spraying, leaf disc method, rice embryo transformation method, etc.

[0094] Plant modification

[0095] Through a large number of systematic research and large-scale research screening, the present application identifies the ERF04 gene, which regulates the traits of seed dormancy and seed germination of plants. Up-regulation of the ERF04 gene can reduce the seed dormancy of plants or promote germination, and down-regulation of the ERF04 gene can increase the seed dormancy of plants or reduce germination.

[0096] Based on the new findings of the present inventors, the present application provides the use of an ERF04 gene, a protein encoded by the ERF04 gene, an ERF04 gene promoter or a modulator thereof for regulating the seed dormancy and seed germination of plants in the family Poaceae.

[0097] It should be understood that after the regulatory effect of the ERF04 gene in plants in the family Poaceae is known, various methods well known to those skilled in the art can be used to regulate the expression or activity of the ERF04 gene or the protein encoded by the ERF04 gene according to the actual needs, which are all included in the present application.

[0098] The activity of ERF04 can be up-regulated using the ERF04 gene, the protein encoded by the ERF04 gene, or an up-regulator of the ERF04 gene promoter. The ERF04 gene, the protein encoded by the ERF04 gene, or the up-regulator of the ERF04 gene promoter includes a promoter, an agonist, and an activator. The "up-regulation" and "promotion" include "up-regulation" and "promotion" of the activity of the protein or "up-regulation" and "promotion" of the expression of the protein. Any substance that can increase the activity of the ERF04 protein, increase the stability of the ERF04 gene or the ERF04 protein, up-regulate the expression of the ERF04 gene or the ERF04 gene promoter, or increase the effective time of the ERF04 protein can be used in the present application as a substance useful for up-regulating the ERF04 gene or the protein encoded by the ERF04 gene. The substance can be a compound, a chemical small molecule, or a biological molecule. The biological molecule can be at the nucleic acid level (including DNA and RNA) or at the protein level.

[0099] As a preferred embodiment, a method for up-regulating the expression or activity of the ERF04 gene or the protein encoded by the ERF04 gene in a plant is provided, and the method includes introducing an expression construct or a vector containing the ERF04 gene into a plant.

[0100] Preferably, a method for preparing a transgenic plant is provided, and the method includes:

[0101] (1) introducing an exogenous nucleic acid encoding ERF04 into a plant organ or tissue to obtain a plant tissue or organ into which the nucleic acid is introduced; and

[0102] (2) regenerating a plant plantlet from the plant tissue or organ into which the exogenous nucleic acid is introduced obtained in step (1).

[0103] Preferably, the method includes the step of:

[0104] (s1) providing Agrobacterium carrying an expression vector containing an exogenous (recombinant) ERF04;

[0105] (s2) contacting a plant tissue or organ with the Agrobacterium in step (s1) so that the ERF04 is introduced into and integrated into the chromosome of a plant cell;

[0106] (s3) selecting a plant cell, tissue, or organ into which the ERF04 is introduced.

[0107] The present application also includes a plant obtained using any of the aforementioned methods, and the plant includes a transgenic plant into which the ERF04 is introduced.

[0108] In the present application, the ERF04 gene, the protein coded by the ERF04 gene, and the down-regulator of the ERF04 gene promoter refer to any substance that can reduce the activity of the ERF04 protein, reduce the stability of the ERF04 gene or the protein coded by the ERF04 gene, down-regulate the expression of the ERF04 gene, the ERF04 gene promoter, reduce the effective action time of the ERF04 protein, inhibit the transcription and translation of the ERF04 gene, or reduce the phosphorylation / activation level of the protein. These substances can be used in the present application as substances useful for down-regulating the ERF04 protein. They can be compounds, chemical small molecules, or biological molecules. The biological molecules can be at the nucleic acid level (including DNA and RNA) or at the protein level.

[0109] For example, the down-regulator is an interfering RNA molecule or an antisense nucleotide that specifically interferes with the expression of the ERF04 protein or other signal pathway genes, or a gene editing reagent that specifically edits the ERF04 gene, etc.

[0110] As a preferred mode of the present application, a method for down-regulating the ERF04 gene in plants is provided, which comprises targeted mutation, gene editing, or gene recombination of the ERF04 gene, so as to achieve down-regulation. As a more specific example, the ERF04 gene is converted into a mutant thereof by any of the above methods, so as to no longer function. As a more specific example, the CRISPR / Cas9 system is used for gene editing. Suitable sgRNA target sites can bring higher gene editing efficiency, so suitable target sites can be designed and found before gene editing. After designing specific target sites, in vitro cell activity screening is also needed to obtain effective target sites for subsequent experiments. Preferred gene editing reagents are provided in the examples of the present application.

[0111] Plant targeted screening and molecular markers

[0112] Based on the new findings of the present inventors, the present application provides a molecular marker suitable for identifying plant traits, i.e., the ERF04 gene; the plant traits are seed dormancy and seed germination. The present application also relates to specific molecular markers designed for the ERF04 gene, and identification strategies.

[0113] As a preferred mode, the method for directional selection or identification of plants with modulated agronomic traits according to the present application comprises: identifying the expression or activity of ERF04 gene or its encoded protein in a test plant or its seeds; if the expression or activity of ERF04 gene or its encoded protein in the test plant or its seeds is higher than the average expression or activity value of the plant or its seeds, the test plant or its seeds is a plant with increased seed dormancy or reduced germination; if the expression or activity of ERF04 gene or its encoded protein in the test plant or its seeds is significantly lower than the average expression or activity value of the plant or its seeds, the test plant or its seeds is a plant with reduced seed dormancy or promoted germination.

[0114] According to the new discovery of the present application, one skilled in the art can use any of the techniques known in the art or being developed to analyze the nucleic acid sequence, which can be included in the present application. The methods include, but are not limited to, sequencing, PCR amplification, probe, hybridization, restriction enzyme analysis, and allele polymorphism analysis (e.g. melting curve method) for identification of nucleic acid sequence.

[0115] The present application has a good application prospect in molecular design breeding and improvement of crop varieties using genetic engineering technology.

[0116] After the function of ERF04 gene is known, it can be used as a molecular marker for directional screening of plants. Based on the new discovery, substances or potential substances that can modulate the mechanism to direct the seed dormancy and germination of plants in the family Poaceae can be screened.

[0117] The present application provides a method for screening modulators of seed dormancy and germination of plants in the family Poaceae, comprising: adding a candidate substance to a system containing ERF04 gene or its encoded protein; detecting the system to observe the expression or activity of ERF04 gene or its encoded protein. If the candidate substance up-regulates ERF04 gene or its encoded protein, it indicates that the candidate substance is a modulator that reduces seed dormancy or promotes germination; if the candidate substance down-regulates the expression or activity of ERF04 gene or its encoded protein, it indicates that the candidate substance is a modulator that increases seed dormancy or reduces germination of plants.

[0118] Methods for screening substances acting on a target using the target as a protein or gene or a specific region thereof are well known to those skilled in the art, and these methods can be used in the present application. The candidate substance can be selected from the group consisting of peptides, poly-peptides, peptidomimetics, non-peptide compounds, carbohydrates, lipids, antibodies or antibody fragments, ligands, small organic molecules, small inorganic molecules, and nucleic acid sequences. Depending on the type of substance to be screened, one skilled in the art knows how to select an appropriate screening method.

[0119] The interaction between proteins and the strength of the interaction can be detected by various techniques well known to those skilled in the art, such as GST pull-down, bimolecular fluorescence complementation, yeast two-hybrid system or immunoprecipitation.

[0120] Through large-scale screening, a class of substances that specifically act on ERF04 gene, the protein encoded by the ERF04 gene, the promoter of the ERF04 gene, and have a regulatory effect on the improvement of the traits of the Poaceae plants can be obtained.

[0121] In order for those skilled in the art to better understand the technical solutions of the present application, the technical solutions in the embodiments of the present application will be described clearly and completely below in combination with the drawings in the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative efforts should fall within the scope of protection of the present application. The experimental methods not specified in the following embodiments are usually according to the conditions described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Third Edition, Science Press, or according to the conditions suggested by the manufacturers.

[0122] The present application provides a new type of gene ERF04 for regulating seed dormancy and germination of plants, further discloses that the deletion of the ERF04 gene leads to the increase of seed dormancy and the reduction of the phenomenon of ear germination, and the overexpression of the ERF04 gene can reduce the seed dormancy. The present application provides a new way for the improvement of seed dormancy and the reduction of ear germination of plants.

[0123] The experimental methods used in the following embodiments are conventional methods unless otherwise specified.

[0124] The materials and reagents used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0125] Example 1: Obtaining of the pericarp and endosperm-specific expression promoter fragment of rice

[0126] (1) Screening of the transcription factor for pericarp and endosperm-specific expression of rice

[0127] The mRNA was extracted from the rice leaf, root, spikelet, rice seed after 4, 7, 11, 14, 21 days and the rice seed starch endosperm and aleurone layer (11-day seed) separated by hand, and the cDNA was synthesized by reverse transcription, and the expression profile of rice OsERF04 gene (gene number LOC_Os08g45110) in different tissues and development periods was identified by real-time fluorescent quantitative PCR. The primer sequence (5'-3') used in the fluorescent quantitative PCR is shown in SEQ ID NO. 1 and SEQ ID NO. 2. The fluorescent quantitative PCR result is shown in Figure 1, and it is preliminarily determined that the OsERF04 gene is specifically expressed in the rice pericarp aleurone layer and embryo.

[0128] (2) Cloning of the promoter fragment of the rice pericarp aleurone and embryo-specific expression gene

[0129] The genomic DNA of rice Zhonghua 11 (hereinafter also referred to as wild type rice; Ni Yuchong, 1989; publicly available from the Institute of Botany, Chinese Academy of Sciences) leaf was extracted as a template for PCR amplification, and the primer sequences are shown in SEQ ID NO. 3 and SEQ ID NO. 4.

[0130] The PCR amplification system is as follows: KOD FX buffer 25 μL, genomic DNA (100 ng / μL) 2 μL, dNTP (25 mM) 1 μL, upstream primer (2 μM) 4 μL, downstream primer (2 μM) 4 μL, KOD FX 0.5 μL, ddH2O 10.5 μL.

[0131] The PCR amplification program is as follows: pre-denaturation 95℃ 2 min, denaturation 98℃ 30 s, annealing 60℃ 30 s, 68℃ extension 160 s, 68℃ 5 min, wherein the denaturation, annealing and extension are 35 cycles in total.

[0132] After sequencing, the size of the PCR product is 2424 bp (containing a 38 bp linker), and the OsERF04 promoter sequence is 2386 bp (without the 38 bp linker sequence) as shown in SEQ ID NO. 5, which is named P-OsERF04.

[0133] Example 2 Obtaining of recombinant bacteria with P-OsERF04

[0134] 1. Obtaining of recombinant vector

[0135] The expression vector pPLV15 (De Rybel B et al., 2011; publicly available from the Institute of Botany, Chinese Academy of Sciences) is digested with Hpa I, and the P-OsERF04 fragment and the Hpa I-digested vector pPLV15 are respectively digested with T4 DNA polymerase to form complementary sticky ends, and then the two digestion products are mixed and ligated. The specific digestion system is as follows:

[0136] (1) The pPLV15 vector is digested with Hpa I: 50 μL of the digestion system includes 1 μg of pPLV15 plasmid, 5 μL of 10×Hpa I buffer, 2 μL of Hpa I, and ddH2O is added to 50 μL. The enzyme is incubated at 37°C for 2 hours, and the linearized pPLV15 of 7432 bp is recovered.

[0137] (2) The linearized pPLV15 vector is digested with T4 DNA polymerase: 50 μL of the digestion system includes 500 ng of the linearized pPLV15 vector, 5 μL of 10×T4 buffer, 5 μL of 100 mM dGTP, 2.5 μL of 100 mM DTT, 0.5 μL of BSA (100X), 1 μL of T4 DNA polymerase, and ddH2O is added to 50 μL. The enzyme is incubated at 22°C for 2 hours, and denaturation is performed at 75°C for 20 minutes to obtain the pPLV15 digestion product of about 7432 bp.

[0138] (3) The P-OsERF04 PCR product is digested with T4 DNA polymerase:

[0139] The digestion system is as follows (20 μL system): 200 ng of the P-OsERF04 fragment, 2 μL of 10×T4 buffer, 2 μL of 100 mM dGTP, 2 μL of 100 mM DTT, 0.2 μL of BSA (100X), 0.4 μL of T4 DNA polymerase, and ddH2O is added to 20 μL. The enzyme is incubated at 22°C for 2 hours, and denaturation is performed at 75°C for 20 minutes to obtain the P-OsERF04 fragment digestion product of about 2424 bp.

[0140] (4) 20 μL of the P-OsERF04 digestion product and 3 μL of the pPLV15 digestion product are ligated at 22°C for 2 hours to obtain the ligation product.

[0141] (5) The ligation product is heat-shocked to transform the DH5α E. coli competent cells to obtain clones, and bacterial liquid PCR identification is performed to obtain a positive clone. The plasmid is extracted, and the plasmid is named as pPLV15-P-OsERF04, which is the recombinant vector.

[0142] 2. Obtaining of the recombinant bacteria

[0143] The recombinant vector pPLV15-P-OsERF04 was electroporated into Agrobacterium tumefaciens EHA105 (Hood E et al., 1993; publicly available from the Institute of Botany, Chinese Academy of Sciences), and positive clones were identified by bacterial liquid PCR. The target fragment was 2424 bp, and the recombinant bacteria EHA105 / pPLV15-P-OsERF04 were obtained.

[0144] The specific steps for transforming Agrobacterium tumefaciens are as follows:

[0145] 1) 1 μL of the recombinant vector pPLV15-P-OsERF04 and 1 μL of the pSOUP plasmid (Hellens RP et al., 2000; publicly available from the Institute of Botany, Chinese Academy of Sciences) were added to EHA105 Agrobacterium competent cells, and mixed well;

[0146] 2) Incubate on ice for 30 minutes, and prepare the electrode cup at the same time. Wash the electrode cup with anhydrous ethanol and dry it on ice for standby;

[0147] 3) Electroporate at 1800 volts for 6.2 milliseconds;

[0148] 4) Add 1 mL of YEB liquid medium, and activate in an incubator or shaker at 28°C for 2 hours;

[0149] 5) Coated with resistant plates: YEB medium with rifampicin (25 μg / mL) and kanamycin (50 μg / mL) solid medium.

[0150] 6) Incubate in an incubator at 28°C for 48 hours.

[0151] 7) Pick single colonies in YEB liquid medium with rifampicin (25 μg / mL) and kanamycin (50 μg / mL), and incubate in a shaker at 28°C and 200 rpm for 1 day.

[0152] 8) Take 0.5 mL of bacterial liquid and transfer to 50 mL (1 / 100 dilution) of the same YEB resistant medium, and incubate under the same conditions until the OD 600 = 0.5 or so, and then proceed to callus transformation.

[0153] Example 3: Obtaining and molecular identification of P-OsERF04 rice transgenic plants

[0154] The recombinant bacteria EHA105 / pPLV15-P-OsERF04 were transformed into rice Zhonghua 11 callus, and T0 generation P-OsERF04 rice was obtained.

[0155] The specific experimental procedures for transformation are as follows:

[0156] (1) Seed sterilization: The seeds of rice Zhonghua 11 were sterilized by removing the hulls and immersing in 70% ethanol for 10 min, and then in 0.1% HgCl2 solution for 10 min. The seeds were washed with sterile water for 10 times. After 16 hours, the extra water was removed, but not too dry;

[0157] (2) Inoculation and induction of callus: The seeds were cut into pieces on sterile filter paper, and the pieces were placed on induction medium NB2 with the scutellum facing up. Each 13*13 cm square dish was inoculated with 40 pieces. The callus was induced to form by dark culture at 25°C for 4 weeks;

[0158] (3) Subculture: The induced callus was transferred to subculture medium NB1 and cultured in a 25°C incubator for 2 weeks under 24-hour dark condition (not more than 4 weeks). The light yellow and dense embryogenic callus was transferred to new NB1 subculture medium for continuous culture for 2 weeks;

[0159] (4) Pre-culture of callus to be transformed: The yellow and white dense embryogenic callus with good growth was selected, cut into 2 mm pieces, and transferred to new NB1 medium for dark culture at 25°C for 4 days;

[0160] (5) Activation culture of Agrobacterium: The Agrobacterium EHA105 carrying the transformation plasmid was streaked and cultured. A single colony was picked and inoculated in 10 mL YEB (containing Kan 50 μg / mL and Rif 25 μg / mL) and cultured at 28°C with shaking until the logarithmic phase (OD 600 = 0.8). Then, 0.5 mL of the culture was transferred to 50 mL (1 / 100 dilution) of the same medium, and cultured under the same conditions until OD 600 = 0.5;

[0161] (6) Co-culture and transformation: The activated Agrobacterium was centrifuged at 4200 g for 10 min, and the medium was discarded. The bacterial cells were resuspended in an equal volume of AAM-AS medium. The callus pieces (cut into 2 mm pieces) pre-cultured for 4 days were collected in a small beaker, immersed in the AAM-AS bacterial solution for 20 min, and then the bacterial solution was removed and the callus pieces were dried with a pipette. Then, the callus pieces were placed in a dish covered with 4 layers of filter paper and dried for about 30 min. Finally, the callus pieces were transferred to NB2C co-culture medium covered with a layer of filter paper, and co-cultured in the dark at 25°C for 2 days;

[0162] (7) Removal of Agrobacterium after co-culture and selection culture: The callus pieces cultured on NB2C medium were transferred to NBS1 selection medium and cultured in the dark for two weeks. After two weeks, the callus pieces were transferred to NBS2 selection medium and cultured in the dark for two generations, each generation for 15 days. Finally, the fresh yellow hygromycin-resistant callus was obtained;

[0163] (8) Differentiation culture: the resistant callus was transferred to the pre-differentiation medium RE1 for differentiation culture, first 25°C dark culture for 2 weeks, then 25°C culture under light for 2 weeks, the callus which had differentiated into adventitious buds was transferred to the differentiation medium RE2 for light culture for 2 weeks, if the state was not good, it could be subcultured on RE2 again; the undifferentiated callus block was treated by second time partial drying, and pre-differentiation and differentiation on RE1 and RE2 again, and the regeneration plantlet yield could reach 100%;

[0164] (9) The differentiated plantlet was transferred to the rooting medium containing 50 mg / L hygromycin when it grew to 2 cm high. The rooting and strong seedling culture was carried out on the rooting medium at 25°C under 12 hours light;

[0165] (10) When the root system grew well and the seedling grew to 8 cm, the cover was opened for 6 days. The tissue culture seedling was taken out and transferred to the soil, placed in the greenhouse without direct sunlight for acclimation culture, and then moved to outdoor cultivation. The TO generation of P-OsERF04 rice was obtained.

[0166] Preparation of main culture media for rice transformation:

[0167] 1) Preparation of induction medium and subculture medium

[0168] 2) NB2C co-culture medium: NB2 medium, 10 g / L glucose, 100 μmol / L acetyl-syringone (As), 7 g / L agar, adjust pH to 5.2.

[0169] 3) Preparation of AAM-As transformation medium

[0170] 4) Preparation of NBS1 screening medium

[0171] 500 mL NB1 medium (pH 5.8) was added with 3.5 g agar, after sterilization, when it was cooled to 50°C, 250 μL of 50 mg / mL hygromycin Hyg B (final concentration 25 mg / L) and 1125 μL of TMT 100 mg / mL (225 mg / L) were added.

[0172] 5) Preparation of NBS2 screening medium

[0173] 500 mL NB1 medium (pH 5.8) was added with 3.5 g agar, after sterilization, when it was cooled to 50°C, 500 μL of 50 mg / mL hygromycin Hyg B (final concentration 50 mg / L) and 900 μL of TMT 100 mg / mL (180 mg / L) were added.

[0174] 6) Preparation of RE1 / RE2

[0175] 7) Preparation of rooting medium: ½ MS + NAA (0.2 mg / mL, or without)

[0176] 8) Preparation of medium stock solution

[0177] N6 macro (10x):

[0178] B5 micro (1000x):

[0179] B5 organic (100x):

[0180] Iron salt (100x):

[0181] N6 calcium salt (10x): CaCl2.2H2O 1.66 g dissolved in 100 mL water.

[0182] Myo-inositol (200x): 4 g dissolved in 200 mL (20 mg / mL).

[0183] MS macro (20x):

[0184] MS micro (1000x):

[0185] MS organic: glycine 0.4 g, VB1 0.08 g, VB6 0.1 g, nicotinic acid (VB5) 0.1 g; MS calcium salt (100x): 4.4 g CaCl2.2H2O dissolved in 100 mL water.

[0186] Preparation of hormones:

[0187] 2,4-D (1 mg / mL): first dissolved in a small amount of anhydrous ethanol or 95% ethanol, then diluted with water to volume. 0.1 g dissolved in 100 mL water.

[0188] 6-BA (1 mg / mL): first dissolved in 1 M HCl, then diluted to volume. 0.1 g dissolved in 100 mL water.

[0189] ZT (zeatin, 1 mg / mL): first dissolved in 95% ethanol with heating, then diluted to volume with water.

[0190] KT (kinetin, 0.5 mg / mL): 0.05 g KT first dissolved in a small amount of 1 M HCl, then diluted to 100 mL with water.

[0191] NAA (Naphthaleneacetic acid, 0.5 mg / mL): 0.05 g NAA was dissolved in a small amount of 95% ethanol, and then water was added to 100 mL.

[0192] AAM-AS bulk (10x):

[0193] AAM-AS organic (1000x):

[0194] AAM-AS amino acids (10x):

[0195] AS (Aryloxybenzylisonitrile) preparation: Mr: 196.2, stock concentration: 50 mM / L;

[0196] TMT (TMT) preparation: 1.6 mg / bottle TMT plus 14 mL sterilized water, after complete dissolution, filter sterilization and sub-packaging into sterilized centrifuge tubes.

[0197] Note: Only AS (Aryloxybenzylisonitrile) and TMT are filter sterilized, and other hormones can be autoclaved.

[0198] 2. Identification of transgenic plants

[0199] The T0 generation of transgenic P-OsERF04 rice was transplanted into an artificial climate chamber at 30°C / 28°C with 16h light / 8h dark, and genomic DNA was extracted from the leaves. The presence of the exogenous gene and its integration into the genome were identified using a specific primer for hygromycin (the hygromycin coding gene is on the pPLV15 vector). The PCR amplification primer sequences (5'-3') are shown in SEQ ID NO. 6 and SEQ ID NO. 7.

[0200] The results obtained a 501 bp PCR product of interest, which was a positive T0 generation of transgenic P-OsERF04 rice.

[0201] Example 4 GUS staining of transgenic P-OsERF04 rice to verify promoter function

[0202] The T1 generation of transgenic P-OsERF04 positive rice was removed from the inner and outer glume at the grain filling stage (4, 7, 11, 14, 21, 28 days after flowering), and then the seeds were cut horizontally or vertically. The horizontally or vertically cut seeds were placed in an acetone solution, which was pre-cooled on ice, and then vacuumed for 10 minutes before placing the material in a -20°C refrigerator for 1 h. The acetone was discarded, and 500 μL of GUS staining solution without X-gluc was used to elute twice, and then GUS staining solution with X-gluc was added, and placed in a 37°C incubator for 1-12 hours. Whether GUS signal appeared was observed under a stereomicroscope and photographed.

[0203] GUS staining solution: Phosphate buffer (pH 7) 0.1 M, EDTA solution (pH 8) 0.01 M, K + ferricyanide solution 2 mM, K + ferrocyanide solution 2 mM, Triton X-100 1 μL / mL, X-gluc 0.5 μg / μL.

[0204] The results of T1 generation of P-OsERF04 transgenic rice are shown in Figure 2. The GUS gene of T1 generation of P-OsERF04 transgenic rice has expression activity in the aleurone layer and embryo of the caryopsis (blue), and does not express in other parts of the caryopsis. In addition, it does not express in the leaf, leaf sheath, cross section of node, inter-node and outer sheath of the inflorescence axis, floret, stamen and pistil.

[0205] The results show that P-OsERF04 is a promoter that can drive specific expression of a target gene such as GUS reporter gene in the aleurone layer and embryo of the caryopsis.

[0206] Example 5 Obtaining of OsERF04-RNAi transgenic lines and analysis of grain dormancy phenotype

[0207] 1. Construction of pUBi-OsERF04-RNAi vector:

[0208] According to the sequence of the coding region of OsERF04 gene, forward and reverse primers were designed (the primer sequences are shown in SEQ ID NO. 8 and SEQ ID NO. 9), and Kpn I and Spe I enzyme digestion sites were added in front of the forward primer, and BamH I and Sac I enzyme digestion sites were added in front of the reverse primer. The cDNA of Zhonghua 11 caryopsis was used as a template to amplify a partial fragment of the coding region of OsERF04, and the nucleotide sequence is shown in SEQ ID NO. 10. The fragment size is 384 bp and the total size of the 4 enzyme digestion site sequences is 408 bp.

[0209] The specific steps of vector construction are as follows:

[0210] 1) Enzymatic digestion of PCR gel-recovered product (408 bp) and pTCK303 vector (Wang Zhen et al., 2004; publicly available from the Institute of Botany, Chinese Academy of Sciences):

[0211] BamH I and Sac I enzyme cut PCR cut gel recovery product (408bp), enzyme cutting system is 20 μL: PCR product cut gel recovery product 500 ng, 10 x cutsmart buffer 2 μL, BamH I 1 μL, Sac I 1 μL, ddH2O is added to 20 μL.

[0212] BamH I and Sac I enzyme cut pTCK303 vector, enzyme cutting system is 20 μL: pTCK303 plasmid 1 μg, 10 x cutsmart buffer 1 μL, BamH I 1 μL, Sac I 1 μL, ddH2O is added to 20 μL. 37℃ enzyme cutting 2 hours, recovery linearized pTCK303 vector fragment.

[0213] 2) using T4 DNA ligase for connection, reaction system is 20 μL: the above-mentioned recovery linearized pTCK303 vector fragment 4 μL and 408bp enzyme cutting recovery product 5 μL, and 2 μL of T4 DNA ligase buffer, 1 μL T4 DNA ligase is added, 8 μL of ddH2O is added to 20 μL reaction system, 16℃ connection 2 hours, and the connection product is obtained.

[0214] 3) the above-mentioned connection product is heat shock transformed DH5 alpha E. coli competent cell, and the clone is obtained. The positive clone is identified by broth PCR, and the plasmid of the positive clone is extracted. The results show that the plasmid is the OsERF04 fragment shown in SEQ ID NO. 10 inserted into the pTCK303 vector reversely, and the insertion position is behind the UBI-1 promoter. The plasmid is named as pTCK303-OsERF04 384bp(-) .

[0215] 4) plasmid pTCK303-OsERF04 384bp(-) And 408bp PCR gel recovery product is cut by Spe I-HF and Sac I-HF, and the enzyme cutting system is 20 μL.

[0216] Wherein pTCK303-OsERF04 384bp(-) Is 1 μg, 408bp gel recovery product 500 ng, the amount of Spe I-HF and Sac I-HF is 1 μL, and ddH2O is added to 20 μL. 37℃ enzyme cutting 2 hours, and the 408bp fragment cut by double enzyme cutting and the plasmid pTCK303-OsERF04 384bp(-) .

[0217] 5) T4 DNA ligase for connection, reaction system is 20 μL:

[0218] Linearized pTCK303-OsERF04384bp(-) Carrier fragment 4 μL and 408 bp linearized enzyme cutting recovery product 5 μL, and add 2 μL of T4 DNA ligase buffer, 1 μL of T4 DNA ligase, add 8 μL of ddH2O to 20 μL reaction system; 16 °C for 2 hours, to get the ligation product. The above ligation product was heat shock transformed into DH5α E. coli competent cells, and the clones were obtained.

[0219] The plasmid was double digested with BamH I-HF and Sac I-HF for extracting the plasmid, and a fragment of about 1400 bp was obtained. The plasmid was pTCK303-OsERF04 384bp(-) In the vector, and the insertion position is before the NOS terminator sequence, the plasmid is named pTCK303-OsERF04384 bp (+, -), which is pUBi-OsERF04-RNAi vector.

[0220] 6) The vector was transformed into EHA105 Agrobacterium competent cells according to the method of Example 2, and the recombinant bacteria EHA105 / pUBi-OsERF04-RNAi were obtained; positive clones were identified by bacterial liquid PCR.

[0221] 7) The recombinant bacteria were transformed into Zhonghua 11 rice callus according to the method of Example 3, and transgenic lines were obtained and positive seedlings were identified.

[0222] 8) pUBi-OsERF04-RNAi transgenic line expression detection

[0223] After genotyping the obtained pUBi-OsERF04-RNAi transgenic T1 generation plants, line-28, line-33, line-43 and line-56 were selected as four homozygous lines, and the plants isolated without pUBi-OsERF04-RNAi vector were used as controls. Mature grains were taken to extract RNA, which was reverse transcribed into cDNA, and then the relative expression of OsERF04 was detected by real-time fluorescent quantitative PCR, and the primer sequences are shown in SEQ ID NO. 1 and SEQ ID NO. 2.

[0224] The results are shown in Figure 3, and the expression of OsERF04 in the above four RNAi lines was down-regulated to different degrees.

[0225] Example 6 ERF04-RNAi transgenic line grain dormancy phenotype analysis

[0226] The mature seeds of three of the four transgenic homozygous lines of pUBi-OsERF04-RNAi obtained in Example 5 and the control plants (i.e. wild type) isolated without the vector were subjected to germination experiments.

[0227] The germination rate was counted for 30 consecutive days. It was found that the mature seeds of the three pUBi-OsERF04-RNAi homozygous lines started to germinate gradually 3-4 days after being soaked in water, and some of the seeds just started the germination process 30 days after being soaked in water. However, about 40%-60% of the seeds of the corresponding wild type started to germinate 2-3 days after being soaked in water, and the germination rate reached 100% 5 days after being soaked in water. It can be seen that the mature seeds of the pUBi-OsERF04-RNAi lines showed a delayed germination phenotype (Figures 4A-C). In addition, it was found that, compared with the corresponding wild type, some of the seeds of the OsERF04-kd (OsERF04 knockdown) lines did not start the germination process, and the roots and seedlings of the germinated seeds were significantly shorter (Figures 4D-F).

[0228] Example 7: Obtaining of a rice mutant with enhanced seed dormancy

[0229] CRISPR / Cas9 target site design: The target site design method was referred to Liu Yaoguang's laboratory of South China Agricultural University School of Life Sciences, and two PAM structure sequences NGG were selected near the ATG position of the OsERF04 gene sequence. The 19 nt sequence before GGCA plus NGG was used as the forward primer of the linker, and the reverse complementary sequence was used as the reverse primer. The linker primers of the two target sites were designed.

[0230] The target site and linker sequence are as follows:

[0231] OsERF04-U3-g1-F: see SEQ ID NO. 11;

[0232] OsERF04-U3-g1-R: see SEQ ID NO. 12;

[0233] OsERF04-U3-g2-F: see SEQ ID NO. 13;

[0234] OsERF04-U3-g2-R: see SEQ ID NO. 14.

[0235] CRISPR / Cas9 vector construction: The CRISPR / Cas9 vector construction method was from Liu Yaoguang's laboratory of South China Agricultural University School of Life Sciences, and the vector pYLCRISPR / Cas9P ubi-H (also known as pYLCRISPR / Cas9-MH) and pYLsgRNA-OsU3 (Ma Xingliang et al., 2016; the vector is available from the Institute of Botany, Chinese Academy of Sciences).

[0236] The sequences of the primers used are as follows:

[0237] U-F: see SEQ ID NO. 15;

[0238] gRNA-R: see SEQ ID NO. 16;

[0239] B1': see SEQ ID NO. 17;

[0240] BL: see SEQ ID NO. 18.

[0241] The specific operation steps are as follows:

[0242] (1) Target linker preparation

[0243] The forward and reverse linker primers of the target were dissolved in sterilized deionized water to form a 100 μM stock solution, 1 μL (2 μL in total) of each was added to 98 μL of sterilized deionized water to 1 μM. Denature at 95°C for 5 min, and then slowly cool to room temperature.

[0244] (2) Cutting and connecting

[0245] The system (10 μL) includes the following components: Bsa I Buffer (10x) 1 μL, Bsa I (5 U) 0.5 μL, ATP 0.5 μL (final concentration 0.5-1.0 mM), pYLsgRNA-OsU3 plasmid (10 ng / μL) 1 μL, T4 DNA ligase (35 U) 0.1 μL, prepared target linker 1 μL (final concentration 0.05-0.1 μM), ddH2O 5.9 μL; 37°C for 5 min, 20°C for 5 min, 5 cycles.

[0246] (3) Amplification of gRNA expression cassette

[0247] One round of amplification system 15 μL:

[0248] 1) Reaction 1:

[0249] The system is: 1 μL of the cutting and connecting product in (2) above, U-F primer (10 μM) 0.3 μL, target RP primer (10 μM) 0.3 μL, KOD FX 0.3 μL, 2x KOD FX Buffer 7.5 μL, dNTP 3 μL, ddH2O 2.6 μL.

[0250] 2) Reaction 2:

[0251] System: 1 μL of the edge-cutting and edge-connecting product in the above (2), 0.3 μL of target FP primer (10 μM), 0.3 μL of gRNA-R primer (10 μM), 0.3 μL of KOD FX, 7.5 μL of 2x KOD FX Buffer, 3 μL of dNTP, 2.6 μL of ddH2O; 98°C for 15 s, 60°C for 15 s, 68°C for 20 s, 28 cycles. 4 μL was taken for electrophoresis detection, and a band was taken as a success.

[0252] (4) Second round of amplification (50 μL reaction system):

[0253] The primers B1' and BL were mixed to prepare a 10x working solution, each at 1.5 μM. The first round of PCR product was diluted 10 times with H2O, and if the electrophoresis band was too light, it was not diluted, and 1 μL of each was taken as a template. The amplification system was as follows: 1 μL of reaction 1 product, 1 μL of reaction 2 product, 25 μL of 2x KOD FX Buffer,

[0254] 1 μL of KOD FX, 10 μL of dNTP, 5 μL of B1' + BL, 7 μL of ddH2O; the reaction program was 95°C for 10 s, 58°C for 15 s, 68°C for 20 s, 15-20 cycles. 2-3 μL was taken for electrophoresis and the concentration was roughly estimated.

[0255] PCR amplification product purification: the purification kit used was DNA Clean & Concentrator (D4014, ZYMO RESEARCH, USA), and the specific steps were referred to the instruction manual. Finally, the liquid was collected and the concentration was measured.

[0256] Edge-cutting and edge-connecting to the final vector pYLCRISPR / Cas9P ubi -H (pYLCRISPR / Cas9-MH) (reaction system was 15 μL), including: 20-70 ng of the above PCR purified product, 60-80 ng of pYLCRISPR / Cas9P ubi -H plasmid, 1.5 μL of 10x Bsa I Buffer, 0.5 μL of Bsa I (10 U), and ddH2O was added to 15 μL;

[0257] 37°C for 10 min, 1 μL of ATP (10 mM) and 1 μL of T4 DNA ligase (35 U) were added to the enzyme-digested product, mixed well, 37°C for 2 min, 10°C for 3 min, 20°C for 5 min, 10-15 cycles, and finally 37°C for 2 min to end the reaction.

[0258] Transformation of E. coli with the ligation product:

[0259] The ligation product was added to 100 μL of E. coli competent cells DH5α, incubated on ice for 30 min. After 42°C heat shock for 90 s, immediately placed on ice for 2 min, 1 mL of LB medium was added, and cultured at 200 rpm and 37°C for 1 h. Centrifuged, the supernatant was removed to 100 μL of bacterial solution, and plated on LB solid medium with Kan, and cultured at 37°C overnight. The plasmid was extracted, electrophoresed and sequenced to detect.

[0260] Ten colonies were picked on the Kan plate, shaken and the plasmid was extracted. After electrophoresis and sequencing detection, it was determined that the target sequence was on the vector, i.e. OsERF04-gRNA1-U3-pYLCRISPR / Cas9 and OsERF04-gRNA2-U3-pYLCRISPR / Cas9 recombinant plasmids were obtained.

[0261] Obtaining of rice osERF04 mutant:

[0262] OsERF04-gRNA1-U3-pYLCRISPR / Cas9 and OsERF04-gRNA2-U3-pYLCRISPR / Cas9 recombinant plasmids were electroporated into Agrobacterium tumefaciens EHA105 according to the method of Example 3. After successful transformation was confirmed by liquid bacterial PCR, the rice callus was transformed by large-scale shaking.

[0263] Identification of transgenic positive plants:

[0264] The T0 generation rice was transplanted into an artificial climate chamber at 30°C / 28°C, with light for 16 h / dark for 8 h. The genomic DNA of the leaves was extracted, and M13-F and the target adapter downstream primer were used to identify whether the transgenic plants contained the CRISPR vector. Genotype identification primers were designed, and sequencing of the plants containing the vector was used to identify whether the positive plants were edited at the corresponding site of the OsERF04 gene.

[0265] The identification primer was SEQ ID NO. 19:

[0266] OsERF04-F: SEQ ID NO. 19;

[0267] OsERF04-R: SEQ ID NO. 20.

[0268] Two knock-out mutants were finally obtained at two target sites of the gene by PCR amplification and sequencing, named as oserf04-1 and oserf04-2, respectively. Sequence alignment found that, as shown in Figure 5(A), compared with the wild type, the OsERF04 gene sequence in the two mutants was inserted with an A at the fourth base before the PAM structure of target site 1 and lost 4 bases CCTT at the fourth base before the PAM structure of target site 2, respectively. The OsERF04 gene sequence is shown in SEQ ID NO. 21, and the gene mutant sequences are shown in SEQ ID NO. 22 and SEQ ID NO. 23. It is predicted that the transcripts of the two mutant forms are out of frame and terminated prematurely after encoding a certain number of normal amino acids (Figure 5B). The OsERF04 protein is shown in SEQ ID NO. 24, and the protein mutant sequences are shown in SEQ ID NO. 25 and SEQ ID NO. 26.

[0269] Example 8 Difference in seed germination phenotype, germination rate and main field agronomic traits of mutants

[0270] The mature seeds of the two mutants and the corresponding wild type control plants 45 days after pollination were subjected to germination rate statistics, and the specific steps were as follows:

[0271] Three portions of 50 grains each of Zhonghua 11 and mutant oserf04 seeds were taken and placed in culture dishes lined with three layers of filter paper, ddH2O was added to just cover the rice seeds, and the rice was cultured in a rice culture room.

[0272] The culture conditions were 30°C, light for 14h / dark for 10h. The water was changed every day and the number of germinated seeds was counted. The seeds were considered to have germinated successfully when they turned white.

[0273] The statistical results are shown in Figure 6. It was found that oserf04-1 and oserf04-2 exhibited a delayed seed germination phenotype (Figure 6A). Photographs taken of the seeds 6 days after soaking clearly showed that the wild type had completed seedling morphological development, while the mutants had just started germination, and some were still in dormancy and had not started germination (Figure 6B).

[0274] At the same time, the spike of the wild type, oserf04-1 and oserf04-2 plants 45 days after pollination was subjected to germination rate statistical analysis, and the specific steps were as follows: three spikes of the wild type, oserf04-1 and oserf04-2 plants 45 days after pollination were taken and placed in culture containers lined with three layers of filter paper, ddH2O was added to just cover the rice seeds, and the rice was cultured in a rice culture room. The culture conditions were 30°C, light for 14h / dark for 10h. The water was changed every day and the number of germinated seeds was counted. The seeds were considered to have germinated successfully when they turned white.

[0275] The results show that os erf04-1 and os erf04-2 present the phenotype of delayed seed germination (Figure 6C). It can be clearly seen from the photographs of the panicles after 7 days of soaking that most of the seeds on the wild type panicles have germinated, while only a few seeds on the mutant panicles have just started to germinate (Figure 6D). Once the mutant seeds start to germinate, they will enter the normal stage of seedling morphogenesis.

[0276] According to the previous research results, ERF04 is specifically expressed in the aleurone layer and embryo of rice grains, and it is speculated that the mutation of this gene will not affect the change of other agronomic traits of rice. In order to verify this speculation, the os erf04-1 and os erf04-2 mutants and the corresponding wild type plants at the grain filling stage were observed for preliminary agronomic traits (Figure 7A) and statistically analyzed.

[0277] The measurement and statistical analysis methods of each agronomic trait are as follows:

[0278] 1) Measurement method of plant height: measure from the root of the plant to the top of the longest panicle with a ruler. The number of single plants measured for Zhonghua 11, os erf04-1 and os erf04-2 mutants is 20, 20 and 16 respectively, and finally the average value and standard error of plant height are calculated. The number of tillers is counted for the corresponding single plants measured for plant height of Zhonghua 11, os erf04-1 and os erf04-2 mutants, and finally the average value and standard error are calculated.

[0279] 2) Measurement method of panicle length: measure from the panicle collar node to the top of the panicle with a ruler. 20 panicles of Zhonghua 11, os erf04-1 and os erf04-2 mutants are measured respectively, and finally the average value and standard error are calculated.

[0280] 3) Counting method of panicle grain number: 20 panicles of Zhonghua 11, os erf04-1 and os erf04-2 mutants are taken respectively, and the total number of seeds on each panicle is counted, including shriveled seeds, and finally the average value and standard error are calculated.

[0281] 4) Calculation method of seed setting rate: take the panicles corresponding to the counted panicle grain number, count the number of full seeds on the panicles, and the seed setting rate = full seed number / panicle grain number x 100%, and then calculate the average value and standard error of each sample.

[0282] 5) Calculation method of 100-grain weight: take 300 fresh harvested and un-dried seeds of Zhonghua 11, os erf04-1 and os erf04-2 mutants respectively, and then divide them into 3 equal parts, each part containing 100 seeds, and then weigh them with a one-hundredth scale, and calculate the average value and standard error.

[0283] The results are shown in Figure 7. The oserf04-1 mutant had no significant difference in plant height (Figure 7B), tiller number (Figure 7C), spike length (Figure 7D), grain number per spike (Figure 7E), seed setting rate (Figure 7F) and 100-grain weight (Figure 7G) compared with the wild type. The above results preliminarily confirmed that OsERF04 is a specific regulator of rice grain dormancy and germination.

[0284] Example 9 Cloning of wheat TaERF04 gene

[0285] Using the coding sequence of rice OsERF04 gene to perform alignment on the wheat genome database (WheatOmics 1.0, http: / / 202.194.139.32 / ), there are three genes on the first homologous group chromosome, which are named as TaERF04-1A (TraesCS1A02G244800), TaERF04-1B (TraesCS1B02G256000) and TaERF04-1D (TraesCS1D02G244500), respectively, and their amino acid sequence similarity with OsERF04 is 50.97% to 51.72%. The nucleotide sequence of TaERF04-1A is shown in SEQ ID NO. 27, and the amino acid sequence is shown in SEQ ID NO. 30. The nucleotide sequence of TaERF04-1B is shown in SEQ ID NO. 28, and the amino acid sequence is shown in SEQ ID NO. 31. The nucleotide sequence of TaERF04-1C is shown in SEQ ID NO. 29, and the amino acid sequence is shown in SEQ ID NO. 32.

[0286] According to the cDNA sequence and gDNA sequence of the above-mentioned genes in the database, primers were designed using geneious software, and the gDNA sequence and cDNA sequence of TaERF04 were amplified from wheat variety Zhengmai 7698.

[0287] The primer sequences used for amplification are as follows:

[0288] The primers used for gDNA sequence amplification are as follows:

[0289] TaERF04-1AP2F: see SEQ ID NO. 33;

[0290] TaERF04-1AP2R: see SEQ ID NO. 34;

[0291] TaERF04-1BP2F: see SEQ ID NO. 35;

[0292] TaERF04-1BP2R: see SEQ ID NO. 36;

[0293] TaERF04-1 DP2F: see SEQ ID NO. 37;

[0294] TaERF04-1 DP2R: see SEQ ID NO. 38;

[0295] Primers for amplification of cDNA sequences:

[0296] TaERF04-1 AP6F: see SEQ ID NO. 39;

[0297] TaERF04-1 AP6R: see SEQ ID NO. 40;

[0298] TaERF04-1 BP3F: see SEQ ID NO. 41;

[0299] TaERF04-1 BP3R: see SEQ ID NO. 42;

[0300] TaERF04-1 DP3F: see SEQ ID NO. 43;

[0301] TaERF04-1 DP3R: see SEQ ID NO. 44.

[0302] KOD FX high-fidelity DNA polymerase was used for amplification of the cloned and sequenced fragments, and the PCR system was as follows: 25 μL 2x KOD FX buffer, 10 μL dNTP, 1.5 μL of upper and lower primers (10 pmol / μL) each, 1 μL of template DNA, 1 μL of KOD FX, and 10 μL of ddH2O were added successively, and the total volume was 50 μL.

[0303] KOD amplification PCR program: 94°C pre-denaturation for 30 s; 98°C denaturation for 10 s, 68°C annealing and extension for 40 s (adjust the length according to the amplification speed of 1 kb / min), a total of 45 cycles; the amplification product was stored at 4°C.

[0304] Example 10 TaERF04 specifically expresses in wheat pericarp aleurone layer and embryo

[0305] RT-PCR primers were designed according to the SNPs between TaERF04-1A, 1B and 1D cDNA sequences. The cDNA of 3-day-old germinated seedlings of Chinese Spring wheat, aboveground parts, stems, flag leaves, young panicles, pistils, anthers, seeds, aleurone layers and mature pericarps at different times after pollination and different times after soaking were selected as templates for expression level analysis of TaERF04-1A, TaERF04-1B and TaERF04-1D, and the primer sequences were as follows (5'-3'):

[0306] qRTaERF04-1A-F: see SEQ ID NO. 45;

[0307] qRTaERF04-1A-R: see SEQ ID NO. 46;

[0308] qRTaERF04-1B-F: see SEQ ID NO. 47;

[0309] qRTaERF04-1B-R: see SEQ ID NO. 48;

[0310] qRTaERF04-1D-F: see SEQ ID NO. 49;

[0311] qRTaERF04-1D-R: see SEQ ID NO. 50.

[0312] The results are shown in Figure 8, TaERF04 gene was not detected in the expression of 3-day-old seedling roots and aboveground parts, stems, flag leaves, young panicles, pistils, anthers (Figure 8A), and began to express at a high level in the 7th day of the caryopsis, with the highest expression level in the aleurone layer part, high expression in the mixed tissue of embryo, endosperm and seed coat (seed), and no expression in the pericarp part. TaERF04 had the highest expression in mature caryopsis, and the expression level of TaERF04 decreased rapidly after the caryopsis was soaked. In summary, TaERF04 gene does not express in vegetative tissues and is specifically expressed in caryopsis. After the mature caryopsis was inflated, the expression level of TaERF04 decreased rapidly (Figure 8B).

[0313] Example 11 Obtaining and phenotypic analysis of wheat mutants with enhanced grain dormancy

[0314] CRISPR / Cas9 target design: According to the CRISPR-Cas9 technology operation method shared by Professor Liu Yaoguang's team, the target site was designed, and the editing vector pWMB110-spCas9-TaU3-ERF04-Chr1 was constructed. The target primer sequence is as follows (5'-3'):

[0315] gRT1-ERF04Ch1F: see SEQ ID NO. 51;

[0316] gRT1-ERF04Ch1R: see SEQ ID NO. 52;

[0317] gRT2-ERF04Ch1F: see SEQ ID NO. 53;

[0318] gRT2-ERF04Ch1R: see SEQ ID NO. 54.

[0319] CRISPR / Cas9 vector construction: the construction of editing vector includes target annealing, linearized vector fragment preparation, ligation and transformation, sequencing verification of correct clone and plasmid extraction, Agrobacterium transformation, PCR detection and bacteria preservation.

[0320] 1) 22.5 μL of each upstream and downstream primers, 3.15 μL of NEB buffer, and 50 μL of reaction system were prepared; procedure: 95°C for 5 min, 95°C-25°C per 1 min, 70 cycles, and stop at 10°C.

[0321] 2) pWMB110-spCas9-TaU3 vector was linearized by BamH I and Sac I.

[0322] Reaction volume was 50 μL, containing 2 μg of plasmid, 5 μL of NEB buffer, 1 μL of each endonuclease, and ddH2O to make up 50 μL; 37°C in PCR instrument for 4 h;

[0323] 3) 10 μL of ligation system, containing 1 μL of 10×T4 DNA ligase buffer, 1 μL of vector enzyme digestion product, 7 μL of target double-stranded complementary fragment, and 1 μL of T4 DNA ligase (400 U / μL); 22°C incubator for 30 min, 4°C overnight;

[0324] 4) Escherichia coli strain DH5α competent cells were transformed, and the method referred to the DH5α competent instruction of Tiangen. The vector self-ligation product, linearized fragment, and circular reference plasmid were used as controls. The transformation product was coated on LB (kanamycin resistance) plate, and incubated at 37°C overnight;

[0325] 5) Three colonies were picked from each ligation, and 3 mL of bacteria were shaken overnight, and plasmid was extracted according to the plasmid extraction kit instruction of Tiangen. 10 μL of plasmid was sent for sequencing, and the correct clone was preserved. The corresponding bacterial solution was used for Agrobacterium transformation.

[0326] The constructed vector was transformed into Agrobacterium by three-parent hybridization method, and Agrobacterium-mediated callus transformation was used for wheat receptor material.

[0327] The three-parent hybridization method was as follows:

[0328] 1) C58C1 (rifampicin resistance, color slightly red) strain was cultured in 28°C constant temperature incubator for 24 h in advance;

[0329] 2) 2013K auxiliary bacteria (kanamycin resistance) and Escherichia coli containing editing vector were cultured at 37°C overnight (cultured to the second morning from the night of the same day) in advance for 10 h; +

[0330] ​3) Take 100 μL of each of the three bacteria, mix in a 1.5 mL centrifuge tube, centrifuge at 4000 rpm for 2 min, take 250 μL of supernatant and discard, use the remaining supernatant to mix the bacteria by blowing and sucking, and drop all on the LB solid plate without resistance, when the bacterial plaque is almost dry (inverted without moving), seal, and culture at 28°C for 24 h or more;

[0331] 4) Use a loop to streak the bacterial plaque on the LB solid plate with multiple resistance (rifampicin + kanamycin), culture at 28°C for about 32 h until single colonies of appropriate size are grown. Pick single colonies for culture, plasmid digestion detection or PCR detection (about 2 days).

[0332] Obtaining and genotyping of wheat TaERF04 gene mutants:

[0333] (1) Bacterial liquid PCR identification confirms that the transformation is successful, and a large amount of bacteria can be shaken, and the wheat transgenic platform of the Institute of Crop Sciences, Chinese Academy of Agricultural Sciences is commissioned to use Agrobacterium-mediated method to transform pWMB110-spCas9-TaU3-ERF04-Chr1 editing vector (Figure 9) into Zhengmai 7698 immature embryos to edit TaERF04-Chr1.

[0334] (2) Identification of transgenic positive plants:

[0335] Transplant T0 generation wheat to 8°C spring growth interval, light 16 h / dark 8 h, extract leaf genomic DNA, and use M13-F and target adapter downstream primer to identify whether the transgenic plants contain CRISPR vector. Design TaERF04-1A, TaERF04-1B, and TaERF04-1D universal primers for PCR-RE detection, and samples with incomplete cleavage of the target band are edited plants.

[0336] TaERF04-1A, TaERF04-1B, and TaERF04-1D specific primer sequences are as follows (5'-3'):

[0337] T2 target universal primer:

[0338] ERF4T2-F: see SEQ ID NO. 55;

[0339] ERF4T2-R: see SEQ ID NO. 56;

[0340] TaERF04-1A specific primer:

[0341] ERF4T21A-F: see SEQ ID NO. 57;

[0342] ERF4T21A-R: see SEQ ID NO. 58;

[0343] TaERF04-1B specific primers:

[0344] ERF4T21B-F: see SEQ ID NO. 59;

[0345] ERF4T21B-R: see SEQ ID NO. 60;

[0346] TaERF04-1D specific primers:

[0347] ERF4T21D-F: see SEQ ID NO. 61;

[0348] ERF4T21D-R: see SEQ ID NO. 62.

[0349] Both the cloned and sequenced fragments were amplified using KOD FX high-fidelity DNA polymerase, and the positive transgenic plants were detected using FastLong Taq DNA polymerase amplification.

[0350] KOD FX amplification PCR system: 25 μL 2x KOD FX buffer, 10 μL dNTP, 1.5 μL of upper and lower primers (10 pmol / μL) each, 1 μL template DNA, 1 μL KOD FX, 10 μL ddH2O, total volume 50 μL.

[0351] KOD amplification PCR program: 94°C pre-denaturation for 30 s; 98°C denaturation for 10 s, 68°C annealing and extension for 40 s (adjust the length according to the amplification speed of 1 kb / min), a total of 45 cycles; 4°C storage of amplification product.

[0352] The enzyme digestion reaction system was performed according to the instructions of NEB BsrB I (R0102S) and Pvu II (R0151S), 20 μL of PCR product, 1 μL of restriction endonuclease, 5 μL of 10x cutsmart buffer, 24 μL of ddH2O, total volume 50 μL. Placed in PCR instrument 37°C water bath for 15 min, 80°C water bath for 20 min. 2% agarose gel electrophoresis, using large hole comb dotting 20 μL, 90V, electrophoresis for 1 h.

[0353] After PCR amplification, the products were detected by restriction enzyme digestion, and the results are shown in Figure 10. 20 transgenic single plants with editing were identified at target site 2 (no detection at target site 1, no results attached).

[0354] The identification basis is that the amplification bands of the universal primer and the specific primer in the control Zhengmai 7698 are completely cut into two bands, and the amplification bands of the universal primer in other edited plants all have residual bands that are not cut open (the arrow indicates the amplification product band), indicating that the copy gene has been edited. Then the PCR product is sequenced, and the result comparison analysis shows that the Target2 target site occurs different editing types such as base insertion or deletion, mainly base insertion, and A / G / C / T four types are all present (Fig. 11A).

[0355] Finally, three homozygous triple-copy knockout mutants were selected for further work, named taerf04-2, taerf04-9 and taerf04-10. The sequences of the three mutants all have missense mutations or premature termination mutations (Fig. 11B). Taking taerf04-10 as an example, all three copies have amino acid frame shift mutations, 1A copy has an extra T at the 52nd base, which is predicted to terminate translation after 95 amino acids; 1B copy has a C base deletion at the 52nd base, which is predicted to terminate translation after 7 amino acids; and 1D copy has an A base insertion at the 52nd base, which is predicted to terminate translation after 95 amino acids (Fig. 11B).

[0356] The nucleotide sequence of taerf04-2-1A in the mutants taerf04-2, taerf04-9 and taerf04-10 is SEQ ID NO. 63, and the amino acid sequence is SEQ ID NO. 64; the nucleotide sequence of taerf04-2-1B is SEQ ID NO. 65, and the amino acid sequence is SEQ ID NO. 66; the nucleotide sequence of taerf04-2-1D is SEQ ID NO. 67, and the amino acid sequence is SEQ ID NO. 68; the nucleotide sequence of taerf04-9-1A, taerf04-10-1A is SEQ ID NO. 69, and the amino acid sequence is SEQ ID NO. 70; the nucleotide sequence of taerf04-9-1B is SEQ ID NO. 71, and the amino acid sequence is SEQ ID NO. 72; the nucleotide sequence of taerf04-9-1D, taerf04-10-1D is SEQ ID NO. 73, and the amino acid sequence is SEQ ID NO. 74; the nucleotide sequence of taerf04-10-1B is SEQ ID NO. 75, and the amino acid sequence is SEQ ID NO. 76.

[0357] Example 12 Germination test of wheat mutants with enhanced grain dormancy and influence on other agronomic traits

[0358] Germination experiments were performed on mature spike of taerf04-2, taerf04-9 and taerf04-10 mutants and corresponding wild type Zhengmai 7698. The specific experimental steps are as follows:

[0359] The mutants and wild type were matured synchronously, and 3 spikes of Zhengmai 7698 and taerf04-2, taerf04-9 and taerf04-10 mutants were taken, wrapped with toilet paper, then soaked with ddH2O, and placed in a 25°C constant temperature incubator for dark culture for 7 days. The water was changed every day and the photograph was taken 7 days after soaking.

[0360] The observation results are shown in Figure 12(A), and the spikes of taerf04-2, taerf04-9 and taerf04-10 mutants showed a delayed germination phenotype.

[0361] Another germination experiment was performed on mature caryopsis of taerf04-2, taerf04-9 and taerf04-10 mutants and corresponding wild type Zhengmai 7698, and the specific steps are as follows:

[0362] 30 caryopses of Zhengmai 7698 and taerf04-2, taerf04-9 and taerf04-10 mutants were taken, respectively, and placed in a culture dish lined with three layers of filter paper, then ddH2O was added to just cover the wheat caryopsis, and placed in a 25°C constant temperature incubator for dark culture, and the water was changed every day and the photograph was taken 2 days after soaking.

[0363] The results are shown in Figure 12(B), and the photograph of caryopsis 2 days after soaking can clearly show that the wild type Zhengmai 7698 has completed germination, while most of the caryopses of the three mutants are still in dormancy and have not started germination. The germination rate statistics results show that taerf04-2, taerf04-9 and taerf04-10 mutants show a delayed caryopsis germination phenotype (Figure 12C).

[0364] According to the results of previous studies, TaERF04 is specifically expressed in the aleurone layer and embryo of wheat caryopsis, and it is speculated that mutation of the gene will not affect other agronomic traits of wheat. In order to verify this speculation, the plants at the grain filling stage of taerf04-2, taerf04-9 and taerf04-10 mutants and corresponding wild type Zhengmai 7698 were preliminarily statistically analyzed for agronomic traits.

[0365] The results of FIG. 13 show that the taerf04-2, taerf04-9 and taerf04-10 mutants have no significant difference from the wild type in six main agronomic traits, including plant height, total tiller number, effective tiller number, ear length, grain number per ear and grain weight per ear. The above results preliminarily confirm that TaERF04 is a specific regulator in the process of wheat grain dormancy and germination.

[0366] The above merely describes preferred embodiments of the present application, and is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method of modulating seed dormancy and seed germination in grasses, characterized in that, Adjusting the expression or activity of ERF04 gene or its encoded protein in plants, thereby regulating seed dormancy and seed germination of plants.

2. The method of claim 1, wherein, The method is selected from: (i) up-regulating the expression or activity of ERF04 gene or its encoded protein, thereby reducing seed dormancy or promoting germination; (ii) down-regulating the expression or activity of ERF04 gene or its encoded protein, thereby increasing seed dormancy or reducing germination of plants.

3. The method of claim 2, wherein, The down-regulation of the expression or activity of ERF04 gene or its encoded protein includes any one of the following: knocking out or silencing ERF04 gene in plants, inhibiting the activity of ERF04 protein, or editing the promoter of ERF04 gene to reduce the expression of ERF04 gene.

4. The method of claim 2, wherein, The up-regulation of the expression or activity of ERF04 gene or its encoded protein includes any one of the following: (1) transferring the expression construct or vector of ERF04 gene or the promoter of ERF04 gene into plants; (2) transferring the expression construct or vector containing ERF04 gene or the promoter of ERF04 gene into plants.

5. A method of screening for modulators of modulating seed dormancy and seed germination in grasses, characterized in that, The method comprises adding a candidate substance to a system containing ERF04 gene or its encoded protein, and detecting the system to observe the expression or activity of ERF04 gene or its encoded protein. If the candidate substance up-regulates ERF04 gene or its encoded protein, it indicates that the candidate substance is a modulator for reducing seed dormancy or promoting germination; if the candidate substance down-regulates the expression or activity of ERF04 gene or its encoded protein, it indicates that the candidate substance is a modulator for increasing seed dormancy or reducing germination. The dosage form of the modulator is selected from any one or more of the following: solution, emulsion, suspension, powder, foam, paste, granule, and aerosol.

6. The method of claim 5, wherein, The method comprises the following steps: regenerating the following mutant plant tissues or plant cells into plants to obtain the selected Gramineae plants:

7. A method for breeding a plant of the family Gramineae, characterized by, (1) ERF04 gene knockdown; (2) ERF04 gene knockout; (3) editing the promoter of ERF04 gene to reduce the expression of ERF04 gene. The method comprises the following steps: planting crops and harvesting the grains of the crops; the expression or activity of ERF04 gene or its encoded protein in the crops is reduced.

8. A method of producing foodstuffs, characterized by, 9. Use of any one of ERF04 gene, its encoded protein, ERF04 gene promoter, and modulators thereof in plant breeding. The modulator is ERF04 gene or its encoded protein, expression construct or vector containing ERF04 gene or the promoter of ERF04 gene, for reducing seed dormancy or promoting germination; 10. Use according to claim 9, characterized in that, Or the modulator is a down-regulator of ERF04 gene or its encoded protein, for increasing seed dormancy or reducing germination. The modulator also includes other substances for regulating plant traits.

11. Use according to claim 9, characterized in that, The Gramineae plants are any one or more of the following: rice, wheat, rye, barley, corn, sorghum, and oat.

12. The method of claim 1, wherein, The amino acid sequence of the protein encoded by ERF04 gene is selected from any one of the following:

13. The method of any one of claims 1, 5, 7-8 or use of claim 9, wherein, ​ (a) any one of SEQ ID NO. 24, SEQ ID NO. 30, SEQ ID NO. 31, SEQ ID NO. 32; (b) a protein derived from (a) by substitution, deletion, and / or addition of one or more amino acid residues in the amino acid sequence of any one of SEQ ID NO. 24, SEQ ID NO. 30, SEQ ID NO. 31, SEQ ID NO. 32, and having the function of the protein of (a); (c) a protein having a tag sequence added to the N or C terminus of the amino acid sequence of any one of SEQ ID NO. 24, SEQ ID NO. 30, SEQ ID NO. 31, SEQ ID NO. 32, or a protein having a signal peptide sequence added to the N terminus thereof; (d) a polypeptide having a homology of > 80% to the amino acid sequence of any one of SEQ ID NO. 24, SEQ ID NO. 30, SEQ ID NO. 31, SEQ ID NO. 32, and having the activity of ERF04.

14. The method of any one of claims 1, 5, 7-8 or use of claim 9, wherein, The nucleotide sequence of the ERF04 gene is selected from any one of the following: (i) a polynucleotide encoding a polypeptide as set forth in any one of SEQ ID NO. 24, SEQ ID NO. 30, SEQ ID NO. 31, or SEQ ID NO. 32; (ii) a polynucleotide having a sequence as set forth in any one of SEQ ID NO. 21, SEQ ID NO. 27, SEQ ID NO. 28, or SEQ ID NO. 29; (iii) a polynucleotide having a nucleotide sequence with a homology of > 75% to the sequence as set forth in any one of SEQ ID NO. 21, SEQ ID NO. 27, SEQ ID NO. 28, or SEQ ID NO. 29; (iiii) a polynucleotide having 1 to 60 nucleotides truncated from the 5' end and / or 3' end of the polynucleotide of (ii); (iiiii) a polynucleotide complementary to the polynucleotide of any one of (i) to (iiii).

15. The method according to any one of claims 3 to 4, claim 7 or the use according to any one of claims 9 to 10, characterized in that, The nucleotide sequence of the ERF04 gene promoter is selected from any one of the following: (1) the nucleotide sequence as set forth in SEQ ID NO. 5; (2) a nucleotide sequence having a homology of > 75% to the sequence as set forth in SEQ ID NO. 5.

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