Gene regulating nitrogen fertilizer utilization rate, yield and grain appearance quality of crops, and use thereof

Abstract

A gene regulating the nitrogen fertilizer utilization rate, yield and grain appearance quality of crops, and a use thereof. The involved gene DABE1 and a homologous gene thereof are key regulatory genes for nitrogen response during rice growth and development and participate in the regulation of the stability of G protein γ subunit DEP1 protein. A loss-of-function mutation in the DABE1 and the homologous gene thereof can increase the number of grains of rice per panicle and the grain weight, increase the yield of rice under different nitrogen fertilizer levels, especially under low-nitrogen conditions, and improve the appearance quality of rice grains in a plurality of aspects, thus achieving a synergistic improvement in rice quality, high yield, and efficient nitrogen fertilizer utilization. The DAB1 and the homologous gene thereof have important application value for high-yield, nitrogen-efficient and high-quality breeding of crops.

Description

Genes regulating nitrogen fertilizer use efficiency, yield and grain appearance quality in crops and their applications Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to genes that regulate nitrogen fertilizer utilization efficiency, yield, and grain appearance quality in crops, and their applications. The invention also relates to the amino acid sequence encoded by the gene, as well as methods for cultivating crops with high nitrogen fertilizer utilization efficiency, high yield, and excellent grain appearance quality. Background Art

[0002] Since the 1960s, the first "Green Revolution," characterized by semi-dwarf breeding, has increased the harvest index of rice and wheat, addressing the problems of plant lodging and yield reduction caused by excessive fertilization, thereby significantly increasing rice and wheat yields per unit area. To this day, these semi-dwarf varieties continue to play a vital role in agricultural production. However, for a long time, these varieties, bred under high-fertilizer conditions, have shown a weakened response to nitrogen fertilizer, resulting in a decline in nitrogen use efficiency (NUE). Continuous high-intensity nitrogen fertilizer inputs have led to a series of ecological and environmental pollution problems. Therefore, how to synergistically improve the yield and NUE of these semi-dwarf varieties has become a major issue that needs to be addressed to ensure national food security and sustainable agricultural development (Masclaux-Daubresse et al., 2010).

[0003] Rice yield is a complex quantitative trait controlled by multiple genes and the environment. In recent years, with the rapid development of plant molecular genetics and bioinformatics, multiple key genes for high rice yield and nitrogen efficiency have been identified using techniques such as GWAS analysis, QTL mapping, and map-based cloning. This has laid a foundation for understanding the molecular mechanisms of nitrogen response in rice growth, development, and yield formation, and for breeding nitrogen-efficient, high-yield rice varieties.

[0004] Mutations in the SD1 gene, encoding gibberellin synthase, in Green Revolution rice varieties reduce the amount of active gibberellin in the rice plant, leading to the accumulation of DELLA proteins, key growth repressors in the gibberellin signaling pathway. This reduces plant height and improves lodging resistance under high-fertilizer conditions (Sasaki et al., 2002; Zhang, 2007). The growth regulator GRF4 (GROWTH-REGULATING FACTOR 4) is a key factor in regulating carbon and nitrogen metabolic balance. It forms a complex with GIF1 (GRF-INTERACTING FACTOR 1) to activate the transcription of multiple genes involved in carbon and nitrogen assimilation. Accumulation of DELLA proteins in Green Revolution varieties inhibits the formation of the GRF4-GIF1 complex, resulting in reduced nitrogen absorption and utilization efficiency in rice (Li et al., 2018). The APETALA2 domain-containing transcription factor NGR5 (NITROGEN-MEDIATED TILLER GROWTH RESPONSE 5) is a key player in plant growth and development responses to soil nitrogen levels. NGR5 recruits the PRC2 (POLYCOMB REPRESSIVE COMPLEX 2) complex to methylate histone H3K27me3 on tillering repressor genes, repressing their expression and thereby promoting tillering. Like DELLA proteins, NGR5 is degraded by the 26S proteasome, mediated by the gibberellin receptor GID1. DELLA proteins interact with NGR5 and competitively inhibit the GID1-NGR5 interaction, protecting NGR5 from degradation and promoting tillering (Wu et al., 2020). In "Green Revolution" varieties, simultaneously increasing GRF4 and NGR5 protein levels can improve nitrogen absorption and assimilation capacity while partially uncoupling the coupling effect between tiller number and nitrogen fertilizer, thereby achieving reduced nitrogen fertilizer intake and increased yield. The transcription factor OsTCP19 is involved in regulating nitrogen use efficiency in rice. A 29-bp indel in the promoter region of the OsTCP19 gene results in differences in the response of rice germplasm to nitrogen levels. Rice germplasm carrying OsTCP19-H originates from areas with relatively nitrogen-deficient soils, while rice varieties carrying OsTCP19-L are more common in areas with relatively nitrogen-abundant soils. Incorporating the TCP19-H gene can increase tiller number in rice under low nitrogen conditions, thereby improving nitrogen use efficiency (Liu et al., 2021).

[0005] DEP1 encodes a plant-specific atypical G protein γ subunit (Huang et al., 2009). Rice contains three members of the atypical G protein γ subunit family: GS3, DEP1, and GCC2 (G protein γ subunit type C2). G protein γ subunits contain a G protein γ subunit-like domain (GGL) at their N-termini, which interacts with G protein β subunits to form heterodimers. Compared to classical G protein γ subunits, atypical G protein γ subunits contain a cysteine-rich region at their C-termini (Xu et al., 2016). Studies have shown that DEP1 is a key gene that coordinately regulates rice yield and nitrogen use efficiency. dep1-1 is a semi-dominant mutant allele of DEP1. This mutant replaces a 637-bp fragment in the middle of exon 5 with a 12-bp fragment, generating a stop codon and resulting in a 230-amino acid deletion at the C-terminus. dep1-1 increases grain number per panicle and yield, making it a key high-yield gene. Furthermore, dep1-1 enhances rice's nitrogen absorption and assimilation capacity, ultimately increasing the harvest index and, under appropriate nitrogen reduction conditions, yield, making it a key gene for efficient nitrogen fertilizer utilization (Huang et al., 2009; Sun et al., 2014). However, the molecular mechanism by which dep1-1 responds to nitrogen signals and synergistically improves nitrogen fertilizer use efficiency and yield in rice remains largely unexplored.

[0006] Summary of the Invention

[0007] Using the yeast two-hybrid (Y2H) method, the inventors screened for a protein, DABE1 (DEP1-associated protein 1), that interacts with DEP1. It is an E3 ubiquitin ligase containing a BTB domain. Furthermore, the inventors demonstrated that transcription of the DABE1 gene is regulated by soil nitrogen levels and is induced under low-nitrogen conditions. Further, through phenotypic analysis of near-isogenic rice lines and genetic complementation experiments, the inventors demonstrated that the DABE1 gene regulates nitrogen fertilizer use efficiency, yield, and rice appearance quality. The inventors also found that knocking out DABE1 or multiple knockouts of DABE1 and its homologous genes, DABE2 and DABE3, in rice can increase grain number per panicle and yield under low-nitrogen conditions, achieving both fertilizer reduction and efficiency gains, and improving rice appearance quality. The analysis of the DABE1-DEP1 molecular module lays the theoretical foundation and provides technical support for the synergistic improvement of rice yield and appearance quality.

[0008] The inventors' research will reveal the genetic regulatory network of rice grain number per panicle in response to nitrogen, providing a theoretical basis for molecular design breeding of high-yield and high-quality crops including rice, as well as new gene resources with breeding utilization value.

[0009] Therefore, in general, the present invention provides genes and their applications for regulating nitrogen fertilizer use efficiency, yield, and grain appearance quality in crops. Specifically, the present invention relates to the application of DABE1 and its homologous genes for synergistically improving nitrogen fertilizer use efficiency, yield, and grain appearance quality in rice. The present invention aims to provide important functional genes that can simultaneously improve nitrogen fertilizer use efficiency, yield, and grain quality in crops (e.g., rice, wheat, etc.), as well as methods for cultivating crops with high nitrogen fertilizer use efficiency, high yield, and excellent grain appearance quality.

[0010] In a first aspect, the present invention provides the amino acid sequence of the protein encoded by the gene DABE1 and its homologous genes DABE2 and DABE3 that regulate nitrogen fertilizer utilization efficiency, yield and grain appearance quality of crops.

[0011] In one embodiment, a gene DABE1 is provided for regulating nitrogen fertilizer use efficiency, yield, and rice appearance quality of crops, wherein the amino acid sequence of the protein encoded by the gene DABE1 comprises one selected from the following amino acid sequences:

[0012] 1) the amino acid sequence shown in SEQ ID NO: 2;

[0013] 2) an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 2 by substitution, deletion, and / or insertion of one or more (e.g., 1-25, 1-20, 1-15, 1-10, 1-5, 1-3) amino acid residues, but has the same activity as the protein consisting of the amino acid sequence of SEQ ID NO: 2;

[0014] 3) an amino acid sequence that is at least 70%, preferably at least 80%, more preferably at least 90%, and in particular at least 95%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 2, and has the same activity as the protein consisting of the amino acid sequence of SEQ ID NO: 2;

[0015] 4) An active fragment comprising any one of the amino acid sequences described in 1) to 3).

[0016] In one embodiment, a homologous gene DABE2 of the gene DABE1 that regulates nitrogen fertilizer use efficiency, yield, and rice appearance quality of crops is provided, wherein the amino acid sequence of the protein encoded by the DABE2 gene comprises one selected from the following amino acid sequences:

[0017] 1) the amino acid sequence shown in SEQ ID NO: 4;

[0018] 2) an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 4 by substitution, deletion, and / or insertion of one or more (e.g., 1-25, 1-20, 1-15, 1-10, 1-5, 1-3) amino acid residues, but has the same activity as the protein consisting of the amino acid sequence of SEQ ID NO: 4;

[0019] 3) an amino acid sequence that is at least 70%, preferably at least 80%, more preferably at least 90%, and in particular at least 95%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 4, and has the same activity as the protein consisting of the amino acid sequence of SEQ ID NO: 4;

[0020] 4) An active fragment comprising any one of the amino acid sequences described in 1) to 3).

[0021] In one embodiment, a homologous gene DABE3 of the gene DABE1 that regulates nitrogen fertilizer use efficiency, yield, and rice appearance quality of crops is provided, wherein the amino acid sequence of the protein encoded by the DABE3 gene comprises one selected from the following amino acid sequences:

[0022] 1) the amino acid sequence shown in SEQ ID NO: 6;

[0023] 2) an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 2 by substitution, deletion, and / or insertion of one or more (e.g., 1-25, 1-20, 1-15, 1-10, 1-5, 1-3) amino acid residues, but has the same activity as the protein consisting of the amino acid sequence of SEQ ID NO: 6;

[0024] 3) an amino acid sequence that is at least 70%, preferably at least 80%, more preferably at least 90%, and in particular at least 95%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 6, and has the same activity as the protein consisting of the amino acid sequence of SEQ ID NO: 6;

[0025] 4) An active fragment comprising any one of the amino acid sequences described in 1) to 3).

[0026] The second aspect of the present invention provides the nucleotide sequences of the gene DABE1 and its homologous genes DABE2 and DABE3 that regulate crop nitrogen fertilizer utilization efficiency, yield and grain appearance quality.

[0027] In some embodiments of the present invention, the gene DABE1 for regulating crop nitrogen fertilizer use efficiency, yield and rice appearance quality is provided, wherein the nucleotide sequence of the DABE1 gene comprises one selected from the following:

[0028] 1) the nucleotide sequence shown in SEQ ID NO: 1;

[0029] 2) a nucleotide sequence that differs from the nucleotide sequence shown in SEQ ID NO: 1 by substitution, deletion and / or insertion of one or more (e.g., 1-25, 1-20, 1-15, 1-10, 1-5, 1-3) nucleotide sequences, but the activity of the protein encoded by it is the same as the activity of the protein encoded by the nucleotide sequence shown in SEQ ID NO: 1;

[0030] 3) a nucleotide sequence that is at least 70%, preferably at least 80%, more preferably at least 90%, and especially at least 95%, 98%, or 99% identical to the nucleotide sequence of SEQ ID NO: 1, and that encodes a protein that has the same activity as the protein encoded by the nucleotide sequence of SEQ ID NO: 1;

[0031] 4) nucleotide sequences that differ in sequence from SEQ ID NO: 1 due to the degeneracy of the genetic code;

[0032] 5) An active fragment comprising any one of the nucleotide sequences in 1)-4);

[0033] 6) comprising a nucleotide sequence that hybridizes to a complementary sequence of any one of the nucleotide sequences 1) to 5) under moderately stringent conditions, preferably highly stringent hybridization conditions;

[0034] 7) comprising a nucleotide sequence complementary to any one of the nucleotide sequences in 1) to 5).

[0035] In some embodiments of the present invention, the gene DABE2 for regulating nitrogen fertilizer use efficiency, yield and rice appearance quality of crops is provided, wherein the nucleotide sequence of the DABE2 gene comprises one selected from the following:

[0036] 1) the nucleotide sequence shown in SEQ ID NO: 3;

[0037] 2) a nucleotide sequence that differs from the nucleotide sequence shown in SEQ ID NO: 1 by substitution, deletion and / or insertion of one or more (e.g., 1-25, 1-20, 1-15, 1-10, 1-5, 1-3) nucleotide sequences, but the activity of the protein encoded by it is the same as the activity of the protein encoded by the nucleotide sequence shown in SEQ ID NO: 3;

[0038] 3) a nucleotide sequence that is at least 70%, preferably at least 80%, more preferably at least 90%, and especially at least 95%, 98%, or 99% identical to the nucleotide sequence of SEQ ID NO: 3, and that encodes a protein that has the same activity as the protein encoded by the nucleotide sequence of SEQ ID NO: 3;

[0039] 4) a nucleotide sequence that differs in sequence from SEQ ID NO: 3 due to the degeneracy of the genetic code;

[0040] 5) An active fragment comprising any one of the nucleotide sequences in 1)-4);

[0041] 6) comprising a nucleotide sequence that hybridizes to a complementary sequence of any one of the nucleotide sequences 1) to 5) under moderately stringent conditions, preferably highly stringent hybridization conditions;

[0042] 7) comprising a nucleotide sequence complementary to any one of the nucleotide sequences in 1) to 5).

[0043] In some embodiments of the present invention, the gene DABE3 for regulating crop nitrogen fertilizer use efficiency, yield, and rice appearance quality is provided, wherein the nucleotide sequence of the DABE3 gene comprises one selected from the following:

[0044] 1) the nucleotide sequence shown in SEQ ID NO: 5;

[0045] 2) a nucleotide sequence that differs from the nucleotide sequence shown in SEQ ID NO: 1 by substitution, deletion and / or insertion of one or more (e.g., 1-25, 1-20, 1-15, 1-10, 1-5, 1-3) nucleotide sequences, but the activity of the protein encoded by it is the same as the activity of the protein encoded by the nucleotide sequence shown in SEQ ID NO: 5;

[0046] 3) a nucleotide sequence that is at least 70%, preferably at least 80%, more preferably at least 90%, and especially at least 95%, 98%, or 99% identical to the nucleotide sequence of SEQ ID NO: 5, and that encodes a protein that has the same activity as the protein encoded by the nucleotide sequence of SEQ ID NO: 5;

[0047] 4) a nucleotide sequence that differs in sequence from SEQ ID NO: 5 due to the degeneracy of the genetic code;

[0048] 5) An active fragment comprising any one of the nucleotide sequences in 1)-4);

[0049] 6) comprising a nucleotide sequence that hybridizes to a complementary sequence of any one of the nucleotide sequences 1) to 5) under moderately stringent conditions, preferably highly stringent hybridization conditions;

[0050] 7) comprising a nucleotide sequence complementary to any one of the nucleotide sequences in 1) to 5).

[0051] A third aspect of the present invention provides a method for cultivating crops with high nitrogen fertilizer use efficiency, high yield, and excellent grain appearance quality. The method comprises altering the nucleotide sequence of the gene DABE1, which regulates crop nitrogen fertilizer use efficiency, yield, and grain appearance quality, and its homologous genes DABE2 and DABE3, to produce a functional loss of single or multiple genes, thereby obtaining crops with high nitrogen fertilizer use efficiency, high yield, and excellent grain appearance quality relative to wild-type crops. The method includes, but is not limited to, gene editing technology (e.g., CRISPR-Cas9), and the crops include, but are not limited to, rice.

[0052] In some preferred embodiments, a method for cultivating rice with high nitrogen fertilizer use efficiency, high yield and excellent rice appearance quality is provided. The method uses gene editing technology (such as CRISPR-cas9) to target DABE1 or its homologous genes DABE2 and DABE3, knock out DABE1 or multiple genes knock out DABE1 and its homologous genes DABE2 and DABE3, and obtain rice plants with single gene knockout or multiple gene knockout of DABE1 and its homologous genes, wherein the number of grains per panicle and yield are increased, and the yield and rice appearance quality are synergistically improved under different nitrogen fertilizer levels, especially at low nitrogen levels.

[0053] A fourth aspect of the present invention provides a method for cultivating crops with high nitrogen fertilizer utilization efficiency, high yield and excellent rice appearance quality, the method comprising: hybridizing a crop plant with a single or multiple gene functional loss mutation of the DABE1 gene or its homologous genes with another plant of the crop to obtain a hybrid crop plant, thereby obtaining a crop with high nitrogen fertilizer utilization efficiency, high yield and excellent grain appearance quality relative to the wild type, wherein the crop includes but is not limited to rice.

[0054] In a preferred embodiment, the nucleotide sequences of DABE1 and its homologous genes DABE2 and DABE3 and the amino acid sequences of the proteins encoded therein are shown in SEQ ID NOs: 1-6, see Table 1 below for details.

[0055] Table 1. Sequence names and sources of SEQ ID NOs: 1-6

[0056] The polynucleotides provided by the present invention (i.e., the gene DABE1 or the homologous genes DABE2, DABE3, or other forms of alleles modified by gene editing technology that regulate crop nitrogen fertilizer utilization efficiency, yield and rice appearance quality) or polypeptides are used as targets in improving crop traits (for example, increasing crop yield, nitrogen fertilizer utilization efficiency and / or rice appearance quality).

[0057] The present invention provides uses of DABE1, DABE2 and DABE3, which are used to regulate nitrogen fertilizer utilization rate, yield and / or grain appearance quality of crops, but are not limited thereto.

[0058] The present invention provides a method for breeding improved crop varieties. The method comprises: hybridizing a crop plant containing another allele of DABE1, DABE2, or DABE3 modified using gene editing technology with another crop plant to obtain a progeny crop plant. The obtained crop plant is preferably a crop plant with improved nitrogen use efficiency and yield, and the plant is preferably rice.

[0059] In a more preferred embodiment of the present invention, based on more detailed experimental verification, the inventors have found that the DABE1 gene or its homologous genes DABE2 or DABE3 can be used to cultivate rice varieties with improved nitrogen fertilizer use efficiency, yield and appearance quality through the following two methods:

[0060] (1) Change the gDNA sequence of the DABE1 gene in rice.

[0061] (2) Simultaneously change the gDNA sequences of the DABE1, DABE2, and DABE3 genes in rice.

[0062] Specifically, this application adopts the following technical solutions:

[0063] 1. A protein that regulates nitrogen fertilizer utilization efficiency, yield and / or grain appearance quality of crops, wherein the amino acid sequence is shown in any one of the following:

[0064] 1) amino acid sequences shown in SEQ ID NOs: 2, 4, and 6;

[0065] 2) an amino acid sequence that differs from the amino acid sequence of SEQ ID NOs: 2, 4, or 6 due to substitution, deletion, and / or insertion of one or more amino acid residues, but has the same activity as the protein composed of the amino acid sequence of SEQ ID NOs: 2, 4, or 6;

[0066] 3) an amino acid sequence that is at least 70%, preferably at least 80%, and more preferably at least 90% identical to the amino acid sequence of SEQ ID NO: 2, 4, or 6, and has the same activity as the protein consisting of the amino acid sequence of SEQ ID NO: 2, 4, or 6;

[0067] 4) An active fragment comprising any one of the amino acid sequences described in 1) to 3).

[0068] 2. A gene encoding the protein described in item 1, preferably DABE1 or a homologous gene thereof (e.g., DABE2 or DABE3), the nucleotide sequence of which is shown in any one of the following:

[0069] 1) the nucleotide sequences shown in SEQ ID NOs: 1, 3, and 5;

[0070] 2) A nucleotide sequence that differs from the nucleotide sequence shown in SEQ ID NO: 1, 3, or 5 due to substitution, deletion, and / or insertion of one or more nucleotide sequences, but the activity of the protein encoded by it is the same as that of the protein encoded by the nucleotide sequence shown in SEQ ID NO: 1, 3, or 5;

[0071] 3) a nucleotide sequence that is at least 70%, preferably at least 80%, and more preferably at least 90% identical to the nucleotide sequence of SEQ ID NO: 1, 3, or 5, and that encodes a protein having the same activity as the protein encoded by the nucleotide sequence of SEQ ID NO: 1;

[0072] 4) Nucleotide sequences that differ in sequence from SEQ ID NOs: 1, 3, and 5 due to the degeneracy of the genetic code;

[0073] 5) An active fragment comprising any one of the nucleotide sequences in 1)-4);

[0074] 6) comprising a nucleotide sequence that hybridizes to a complementary sequence of any one of the nucleotide sequences 1) to 5) under moderately stringent conditions, preferably highly stringent hybridization conditions;

[0075] 7) comprising a nucleotide sequence complementary to any one of the nucleotide sequences in 1) to 5).

[0076] 3. Use of a gene knockout vector or gene knockout expression cassette of the gene described in Item 2 in cultivating crops with high nitrogen fertilizer use efficiency, high yield and / or excellent grain appearance quality, wherein the crop is a monocotyledonous plant, preferably rice or wheat, more preferably rice.

[0077] 4. The use according to item 3, wherein the gene knockout expression cassette is an sgRNA expression cassette, and the sequence of the sgRNA expression cassette is shown in SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9.

[0078] 5. A method for cultivating crops with high nitrogen fertilizer use efficiency, high yield and / or excellent grain appearance quality, the method comprising: deleting the function of the gene described in item 2, thereby obtaining a crop with high nitrogen fertilizer use efficiency, high yield and / or excellent grain appearance quality relative to the wild type, preferably, by using the CRISPR-cas9 gene editing method to lose the function of the gene encoding the control of nitrogen fertilizer use efficiency, yield and / or grain appearance quality described in claim 2, thereby obtaining a plant with a single gene knockout of DABE1 or its homologous genes or a plant with multiple gene knockouts of DABE1 and its homologous genes, thereby obtaining a crop with high nitrogen fertilizer use efficiency, high yield and / or excellent grain appearance quality relative to the wild type, wherein the crop is a monocotyledonous plant, preferably rice or wheat, more preferably rice.

[0079] 6. A method for breeding crops with high nitrogen fertilizer utilization efficiency, high yield and / or excellent grain appearance quality, the method comprising: hybridizing a crop plant containing a loss-of-function mutation in the gene described in item 2 with another plant of the crop to obtain a crop with high nitrogen fertilizer utilization efficiency, high yield and / or excellent grain appearance quality relative to the wild type, wherein the crop is a monocotyledonous plant, preferably rice or wheat, more preferably rice.

[0080] 7. The method according to item 6, wherein the crop plant containing the loss-of-function mutation of the gene according to item 2 is obtained by gene editing technology, such as CRISPR-cas9.

[0081] The following are definitions of some terms used in the present invention. Unless otherwise specified, the terms used in the present invention have the meanings known to those skilled in the art.

[0082] In the present invention, “DABE1 gene” and “OsDABE1 gene” both refer to the DABE1 gene of rice.

[0083] "Associated with" / "operably linked" refers to two nucleic acid sequences that are physically or functionally related. For example, a promoter or regulatory DNA sequence is said to be "associated with" a DNA sequence encoding an RNA or protein if the two are operably linked or positioned such that the regulatory DNA sequence affects the expression level of the coding or structural DNA sequence.

[0084] A "chimeric gene" is a recombinant nucleic acid sequence in which a promoter or regulatory nucleic acid sequence is operably linked to, or associated with, a nucleic acid sequence that encodes an mRNA or is expressed as a protein, such that the regulatory nucleic acid sequence regulates the transcription or expression of the associated nucleic acid sequence. The regulatory nucleic acid sequence of a chimeric gene is not normally operably linked to the associated nucleic acid sequence as found in nature.

[0085] A "coding sequence" is a nucleic acid sequence that is transcribed into RNA, such as mRNA, rRNA, tRNA, snRNA, sense RNA or antisense RNA. Preferably, the RNA is then translated in an organism to produce a protein.

[0086] "Hybrid rice" is a general term for first-generation hybrids with hybrid vigor produced by crossing two rice varieties (lines) with different genetic compositions. Currently, three-line hybrid rice and two-line hybrid rice are widely used in production. The production of three-line hybrid rice seeds requires the coordinated development of a male sterile line, a male sterile maintainer line, and a male sterile restorer line. The sterility of the sterile line is controlled by both the cytoplasm and the nucleus, and must be hybridized with the maintainer line to obtain sterile line seeds. The sterile line is then hybridized with the restorer line to obtain hybrid rice seeds for field production. The production of two-line hybrid rice requires only a sterile line and a restorer line. The fertility of the sterile line is regulated by recessive sterility genes within the cell nucleus and by the light intensity and temperature of the growing environment. It undergoes a fertility transition from sterility to fertility with changes in light and temperature conditions, and its fertility is unrelated to the cytoplasm. By utilizing the characteristic of photothermosensitive sterile lines that undergo fertility transitions with changes in light and temperature conditions, seeds can be self-pollinated during suitable light and temperature periods.

[0087] In the context of the present invention, "corresponding to" means that when the nucleic acid coding sequences or amino acid sequences of different DABE1, DABE2, DABE3 genes or proteins are aligned with each other, the nucleic acids or amino acids that "correspond to" certain numerical positions are aligned with these positions, but not necessarily the nucleic acids or amino acids at these exact numerical positions relative to the nucleic acid coding sequence or amino acid sequence of the specific DABE1, DABE2, DABE3, respectively. Similarly, when the coding or amino acid sequence of a specific DABE1, DABE2, DABE3 is aligned with the coding or amino acid sequence of a reference DABE1, DABE2, DABE3, the nucleic acids or amino acids in the specific DABE1, DABE2, DABE3 sequence that "correspond to" certain numerical positions of the reference DABE1, DABE2, DABE3 sequence are aligned with these positions of the reference DABE1, DABE2, DABE3 sequence, but not necessarily the nucleic acids or amino acids at these exact numerical positions of the nucleic acid coding sequence or amino acid sequence of the specific DABE1, DABE2, DABE3 protein, respectively.

[0088] As used herein, an "expression cassette" refers to a nucleic acid sequence capable of directing the expression of a specific nucleotide sequence in a suitable host cell, comprising a promoter operably linked to a nucleotide sequence of interest, the nucleotide sequence of interest being operably linked to a termination signal. Typically, it also comprises sequences required for proper translation of the nucleotide sequence. An expression cassette comprising a nucleotide sequence of interest may be chimeric, meaning that at least one of its components is heterologous to at least one of its other components. An expression cassette may also be naturally occurring but obtained in recombinant form for heterologous expression. However, typically, an expression cassette is heterologous to the host cell, meaning that the specific nucleic acid sequence of the expression cassette does not naturally occur in the host cell and must be introduced into the host cell or a precursor of the host cell through a transformation event. Expression of the nucleotide sequence in the expression cassette may be controlled by a constitutive promoter or an inducible promoter, wherein transcription is initiated only when the host cell is exposed to some specific external stimulus. In the case of multicellular organisms, such as plants, the promoter may also be specific for a particular tissue, organ, or developmental stage.

[0089] A "gene" is a defined region within a genome that, in addition to the aforementioned coding nucleic acid sequence, contains other, primarily regulatory nucleic acid sequences that are responsible for the expression of the coding portion, i.e., transcriptional and translational control. A gene may also contain other 5' and 3' untranslated sequences and termination sequences. Further elements that may be present are, for example, introns.

[0090] A "heterologous" nucleic acid sequence is a nucleic acid sequence that is not naturally associated with the host cell into which it is introduced, and comprises multiple copies of a naturally occurring nucleic acid sequence that is not naturally occurring.

[0091] A "homologous" nucleic acid sequence is a nucleic acid sequence naturally associated with a host cell into which it is introduced.

[0092] An "isolated" nucleic acid molecule or isolated protein is one that has been artificially separated from its natural environment and is therefore not a product of nature. An isolated nucleic acid molecule or protein can exist in a purified form, or can exist in a non-natural environment such as, for example, a recombinant host cell or a transgenic plant.

[0093] "Native gene" refers to a gene found in the genome of an untransformed cell.

[0094] The term "naturally occurring" is used to describe objects that can be found in nature, as opposed to objects that are artificially produced. For example, a protein or nucleotide sequence present in an organism (including a virus) that can be isolated from a natural source and has not been intentionally modified by man in a laboratory is "naturally occurring."

[0095] A "nucleic acid molecule" or "nucleic acid sequence" is a linear fragment of single- or double-stranded DNA or RNA that can be isolated from any source. In the context of the present invention, preferably, a nucleic acid molecule is a DNA fragment. A "nucleic acid molecule" is also called a polynucleotide molecule.

[0096] A "plant" is any plant at any stage of development, in particular a seed plant.

[0097] A "plant cell" is a structural and physiological unit of a plant, comprising a protoplast and a cell wall. A plant cell may be in the form of an isolated single cell or a cultured cell, or as a part of a higher organized unit such as, for example, a plant tissue, a plant organ, or a whole plant.

[0098] "Plant material" refers to leaves, stems, roots, flowers or flower parts, fruits, pollen, egg cells, zygotes, seeds, cuttings, cell or tissue cultures, or any other part or product of a plant.

[0099] A "plant organ" is a distinct and clearly structured and differentiated part of a plant, such as a root, stem, leaf, flower bud or embryo.

[0100] As used herein, "plant tissue" means a group of plant cells organized into a structural and functional unit. This includes any tissue of a plant in planta or in culture. The term includes, but is not limited to, whole plants, plant organs, plant seeds, tissue cultures, and any group of plant cells organized into a structural and / or functional unit. The use of this term in conjunction with any specific type of plant tissue listed above or encompassed by this definition, or by itself, is not intended to exclude any other type of plant tissue.

[0101] A "promoter" is an untranslated DNA sequence upstream of a coding region that contains a binding site for RNA polymerase II and initiates transcription of the DNA. The promoter region may also contain other elements that act as regulators of gene expression.

[0102] "Protoplasts" are isolated plant cells without or with only a partial cell wall.

[0103] "Regulatory elements" refer to sequences involved in controlling the expression of a nucleotide sequence. Regulatory elements include promoters and termination signals operably linked to the nucleotide sequence of interest. They also typically contain sequences required for the proper translation of the nucleotide sequence.

[0104] The phrase "substantially identical" in the context of an alignment of two nucleic acid or protein sequences refers to two or more sequences or subsequences that have at least 60%, preferably 80%, more preferably 90%, even more preferably 95%, and most preferably at least 99% nucleotide or amino acid residue identity when compared and aligned for maximum correspondence, as determined using one of the following sequence comparison algorithms or by visual inspection. Preferably, the substantial identity exists over a region of the sequences that is at least about 50 residues in length, more preferably over a region of at least about 100 residues, and most preferably, the sequences are substantially identical over at least about 150 residues. In particularly preferred embodiments, the sequences are substantially identical over the entire length of the coding region. Furthermore, substantially identical nucleic acid or protein sequences have substantially the same function.

[0105] To perform a sequence comparison, typically, one sequence serves as a reference sequence to which a test sequence is compared. When utilizing a sequence comparison algorithm, the test and reference sequences are input into a computer, subsequence coordinates are specified, if necessary, and sequence algorithm program parameters are specified. The sequence comparison algorithm then calculates the percent sequence identity of the test sequence relative to the reference sequence, based on the selected program parameters.

[0106] Optimal alignment of sequences for comparison can be performed, for example, by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by visual inspection (see generally, Ausubel et al., infra).

[0107] An example of an algorithm suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al., J. Mol. Biol. 215:403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http: / / www.Ncbi.nlm.nih.gov / ). The algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in the search sequence that either match or satisfy some positive threshold score, T, when aligned with a word of the same length in the database sequence. T is called the neighborhood word score threshold (Altschul et al., 1990). These initial neighborhood word hits serve as clues to begin searches to find longer HSPs containing them. These word hits are then extended as far as possible in both directions along each sequence until the cumulative alignment score no longer increases. For nucleotide sequences, the cumulative score is calculated using the parameters M (reward score for pairwise matching residues; always greater than zero) and N (penalty score for mismatching residues; always less than zero). For amino acid sequences, the cumulative score is calculated using a scoring matrix. Extension of word hits in each direction ceases when the cumulative alignment score falls by the number X from the maximum value achieved, when the cumulative score reaches or falls below zero due to the accumulation of one or more negative-scoring residue alignments, or when either sequence reaches an end point. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, a cutoff of 100, M=5, N=-4, and a comparison of both chains. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89: 10915 (1989)).

[0108] In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability that a match between two nucleotide or amino acid sequences would occur by chance. For example, a test nucleic acid sequence is considered similar to a reference sequence if the smallest sum probability compared to the reference nucleic acid sequence is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.

[0109] Another indicator that two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions. The phrase "specifically hybridizes" refers to the ability of a molecule to bind to a specific nucleotide sequence, forming a duplex or hybridizing under stringent conditions, when that sequence is present in a complex mixture (e.g., total cellular DNA or RNA). "Substantial binding" refers to complementary hybridization between the probe nucleic acid and the target nucleic acid, with minimal mismatches that can be tolerated by reducing the stringency of the hybridization medium to achieve the desired detection of the target nucleic acid sequence.

[0110] In the context of nucleic acid hybridization assays such as Southern and Northern hybridizations, "stringent hybridization conditions" and "stringent hybridization wash conditions" are sequence-dependent and differ under different environmental parameters. Longer sequences hybridize specifically at higher temperatures. A wealth of guidance on nucleic acid hybridization can be found in Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Acid Probes, Part I, Chapter 2, "Overview of principles of hybridization and the strategy of nucleic acid probe assays," Elsevier, New York. Generally, high stringency hybridization and wash conditions are selected to be approximately 5°C below the thermal melting point (Tm) for a specific sequence at a defined ionic strength and pH. Typically, under "stringent conditions," a probe will hybridize to its target subsequence but not to other sequences.

[0111] The Tm is the temperature (under defined ionic strength and pH conditions) at which 50% of the target sequence hybridizes to a perfectly matched probe. For a particular probe, very stringent conditions are selected to be equal to the Tm. An example of stringent hybridization conditions for the hybridization of complementary nucleic acids having more than 100 complementary residues on a filter membrane in a Southern or Northern blot is 50% formamide with 1 mg of heparin at 42°C overnight. An example of high stringency wash conditions is 0.15 M NaCl at 72°C for approximately 15 minutes. An example of stringent wash conditions is a 0.2×SSC wash at 65°C for 15 minutes (see, Sambrook, infra, for a description of SSC buffer). Typically, a low stringency wash is performed before a high stringency wash to remove background probe signal. An example of a medium stringency wash for a duplex of, for example, more than 100 nucleotides is a 1×SSC wash at 45°C for 15 minutes. For duplexes of, for example, more than 100 nucleotides, an example of a low stringency wash is a 4-6×SSC wash at 40° C. for 15 minutes. For short probes (e.g., about 10 to 50 nucleotides), stringent conditions typically include a salt concentration of less than about 1.0 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts), at pH 7.0 to 8.3, and typically a temperature of at least about 30° C. Stringent conditions can also be achieved by adding destabilizing agents such as formamide. Generally, a signal-to-noise ratio of 2× (or greater) than that observed for an unrelated probe in a particular hybridization assay indicates detection of specific hybridization. Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the proteins they encode are substantially identical. This occurs, for example, when copies of nucleic acids are created using the maximum codon degeneracy permitted by the genetic code.

[0112] The following are examples of hybridization / washing condition settings that can be used to clone homologous nucleotide sequences that are substantially identical to the reference nucleotide sequences of the present invention: the reference nucleotide sequence is preferably hybridized with the reference nucleotide sequence at 50°C, 7% sodium dodecyl sulfate (SDS), 0.5M NaPO4, 1mM EDTA, and washed at 50°C, 2×SSC, 0.1% SDS, more desirably at 50°C, 7% sodium dodecyl sulfate (SDS), 0.5M NaPO4, 1mM EDTA, and washed at 50°C, 1×SSC, 0.1% SDS, more desirably at 50°C, 7% sodium dodecyl sulfate (SDS), 0.5M NaPO4, 1mM EDTA, and washed at 50°C, 0.5×SSC, 0.1% SDS, preferably at 50°C, 7% sodium dodecyl sulfate (SDS), 0.5M NaPO4, 1mM Hybridize in EDTA and rinse in 0.1×SSC, 0.1% SDS at 50°C, more preferably, hybridize in 7% sodium dodecyl sulfate (SDS), 0.5M NaPO4, 1mM EDTA at 50°C and rinse in 0.1×SSC, 0.1% SDS at 65°C.

[0113] Another indicator that two nucleic acid or amino acid sequences are substantially identical is that the protein encoded by the first nucleic acid immunologically cross-reacts or specifically binds to the protein encoded by the second nucleic acid. Thus, a protein is typically substantially identical to a second protein, for example, where the two proteins differ only by conservative substitutions.

[0114] "Synthetic" refers to a nucleotide sequence that contains structural features not found in natural sequences. For example, an artificial sequence that more closely resembles the G+C content and normal codon distribution of dicot and / or monocot genes is said to be synthetic.

[0115] "Transformation" is the process of introducing heterologous nucleic acid into a host cell or organism. In particular, "transformation" means the stable integration of a DNA molecule into the genome of the organism of interest.

[0116] "Transformed / transgenic / recombinant" refers to a host organism, such as a bacterium or plant, into which a heterologous nucleic acid molecule has been introduced. The nucleic acid molecule can be stably integrated into the host genome or the nucleic acid molecule can exist as an extrachromosomal molecule. Such extrachromosomal molecules can be autonomously replicating. Transformed cells, tissues, or plants are understood to include not only the end product of the transformation process, but also its transgenic progeny. A "non-transformed," "non-transgenic," or "non-recombinant" host refers to a wild-type organism, such as a bacterium or plant, that does not contain the heterologous nucleic acid molecule.

[0117] As used herein, the terms "polynucleotide," "polynucleotide molecule," "polynucleotide sequence," "coding sequence," "open reading frame (ORF)," and the like include single-stranded or double-stranded DNA and RNA molecules that may contain one or more prokaryotic sequences, cDNA sequences, genomic DNA sequences containing exons and introns, chemically synthesized DNA and RNA sequences, and sense and corresponding antisense strands.

[0118] Methods for producing and manipulating the polynucleotide molecules and oligonucleotide molecules disclosed herein are known to those skilled in the art and can be accomplished according to the recombinant techniques described (see Maniatis et al., 1989, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York; Ausubel et al., 1989, Current Techniques in Molecular Biology, Greene Publishing Associates & Wiley Interscience, NY; Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York; Innis et al. (eds.), 1995, PCR Strategies, Academic Press, Inc., San Diego; and Erlich (ed.), 1992, PCR Technology, Oxford University Press, New York).

[0119] "Plant transformation" refers to the expression of at least one exogenous gene in a plant for the purpose of conferring one or more desirable phenotypic traits on the transformed plant.

[0120] In a particularly preferred embodiment, at least one gene for regulating nitrogen fertilizer use efficiency and rice yield according to the present invention is expressed in a higher organism, such as a plant. Specifically, the nucleotide sequence of the gene for regulating nitrogen fertilizer use efficiency and rice yield according to the present invention can be inserted into an expression cassette, and then, preferably, the expression cassette is stably integrated into the plant genome. In another preferred embodiment, the nucleotide sequence of the gene for regulating nitrogen fertilizer use efficiency and rice yield is contained in a non-pathogenic, self-replicating virus and then transfected into plant cells or callus to obtain a transformed plant, also known as a transgenic plant.

[0121] Plants transformed according to the present invention can be monocots or dicots, including but not limited to corn, wheat, barley, rye, sweet potato, bean, pea, chicory, lettuce, cabbage, cauliflower, broccoli, turnip, radish, spinach, asparagus, onion, garlic, pepper, celery, winter squash, pumpkin, hemp, zucchini, apple, pear, quince, melon, plum, cherry, peach, nectarine, apricot, strawberry, grape, raspberry, blackberry, pineapple, avocado, papaya, mango, banana, soybean, tomato, sorghum, sugarcane, sugar beet, sunflower, rapeseed, clover, tobacco, carrot, cotton, alfalfa, rice, potato, eggplant, cucumber, Arabidopsis and woody plants such as conifers and deciduous trees. Particularly preferred are rice, wheat, barley, corn, oats or rye.

[0122] Once a desired nucleotide sequence has been transformed into a particular plant species, it can be propagated within that species or transferred into other varieties of the same species, particularly including commercial varieties, using conventional breeding techniques.

[0123] Preferably, the nucleotide sequences of the present invention are expressed in transgenic plants, thereby causing the biosynthesis of proteins that control nitrogen fertilizer use efficiency and yield traits in the transgenic plants. In this way, transgenic plants with improved traits can be generated. To express the nucleotide sequences of the present invention in transgenic plants, the nucleotide sequences of the present invention may require modification and optimization. All organisms have specific codon usage preferences, as is known in the art, and codon usage can be altered to conform to plant preferences while maintaining the amino acid encoded by the nucleotide sequences of the present invention. Furthermore, high levels of expression in plants are best achieved from coding sequences having a GC content of at least about 35%, preferably greater than about 45%, more preferably greater than 50%, and most preferably greater than about 60%. Although preferred gene sequences can be adequately expressed in both monocot and dicot species, sequences can be modified to accommodate the specific codon and GC content preferences of monocot or dicot plants, as these preferences have been shown to differ (Murray et al., Nucl. Acids Res. 17:477-498 (1989)). In addition, the nucleotide sequences may be screened for the presence of unconventional splice sites that result in message truncation. All changes desired in these nucleotide sequences, such as those described above, may be made using methods described in published patent applications EP 0 385 962 (Monsanto), EP 0 359 472 (Lubrizol) and WO 93 / 07278 (Ciba-Geigy) using site-directed mutagenesis techniques, PCR and synthetic gene construction, as are well known in the art. BRIEF DESCRIPTION OF THE DRAWINGS

[0124] Figure 1. Phenotypic analysis of WYJ7-DEP1, WYJ7-dep1-1 (a mutant in which a 637-bp fragment in the middle of exon 5 is replaced with a 12-bp fragment), the mutant WYJ7-dep1-C1, transgenic plants transformed with pDEP1::DEP1-GFP in the WYJ7-dep1-C1 background (WYJ7-dep1-C1 / pDEP1::DEP1-GFP), and transgenic plants transformed with pDEP1::dep1-1-GFP in the WYJ7-dep1-C1 background (WYJ7-dep1-C1 / pDEP1::dep1-1-GFP) under high and low nitrogen conditions (LN, 120 kg / ha; HN, 300 kg / ha). (A) Plant height. (B) Tiller number. (C) Grain number per ear. Values ​​are means ± SD (n = 15). Different letters in the figure indicate significant differences (P<0.05), and the significance analysis was performed using the Duncan test method.

[0125] Figure 2. Nitrogen responsiveness of agronomic traits of WYJ7-DEP1, WYJ7-dep1-1, WYJ7-dep1-C1, WYJ7-dep1-C1 / pDEP1::DEP1-GFP, and WYJ7-dep1-C1 / pDEP1::dep1-1-GFP. (A) Plant height. (B) Tiller number. (C) Grain number per spike. Values ​​are mean ± SD (n = 15). Different letters indicate significant differences (P < 0.05). Significance analysis was performed using the Duncan test.

[0126] Figure 3. (A) Expression levels of the DEP1 or dep1-1 gene in young panicles of WYJ7-dep1-C1pDEP1::DEP1-GFP and WYJ7-dep1-C1pDEP1::dep1-1-GFP under different nitrogen fertilizer conditions (60 kg / ha, 120 kg / ha, 210 kg / ha, and 300 kg / ha). Values ​​are mean ± SD (n = 3). Different letters indicate significant differences (P < 0.05), and significance was analyzed using the Duncan test. (B) DEP1-GFP or dep1-1-GFP protein accumulation levels in young panicles of WYJ7-dep1-C1 / pDEP1::DEP1-GFP and WYJ7-dep1-C1 / pDEP1::dep1-1-GFP under different nitrogen fertilizer conditions (same as A). HSP82 was used as an internal control for sample loading.

[0127] Figure 4. Statistical analysis of WYJ7-DEP1, WYJ7-dep1-1, WYJ7-dep1-C1, a transgenic line overexpressing the DEP1 gene in the WYJ7-dep1-C1 background (WYJ7-dep1-C1 / pActin::DEP1-GFP), and a transgenic line overexpressing the dep1-1 gene in the WYJ7-dep1-C1 background (WYJ7-dep1-C1 / pActin::dep1-GFP). (A) Plant type, scale bar, 20 cm; (B) Nitrogen response of plant height; (C) Nitrogen response of tiller number; (D) Nitrogen response of grain number per panicle. (B-D) Values ​​in the figures are mean ± SD (n = 15). Different letters indicate significant differences (P < 0.05). Significance analysis was performed using the Duncan test.

[0128] Figure 5. pActin::DEP1-GFP and pActin::dep1-1-GFP transgenic rice materials were cultured under different nitrogen fertilizer conditions (60 kg / ha, 120 kg / ha, 210 kg / ha, and 300 kg / ha), and young panicles were harvested. (A) Transcription levels of DEP1-GFP or dep1-1-GFP in young panicles. Values ​​are mean ± SD (n = 3). OsACTIN1 was used as an internal reference gene. Different letters indicate significant differences (P < 0.05). Significance analysis was performed using the Duncan test. (B) Protein accumulation levels of DEP1-GFP or dep1-1-GFP in young panicles. HSP82 was used as an internal reference gene.

[0129] Figure 6. pActin::DEP1-GFP and pActin::dep1-1-GFP transgenic rice plants were cultured under different nitrogen levels (LN: 0.45 mM NH₄NO₃, HN: 1.5 mM NH₄NO₃). Leaves were obtained after two weeks of cultivation. (A) DEP1-GFP or dep1-1-GFP protein accumulation levels. (B) Ubiquitination levels of DEP1-GFP or dep1-1-GFP proteins. HSP82 was used as an internal control for loading.

[0130] Figure 7. Wild-type WYJ7-DEP1 cells were cultured for two weeks under different nitrogen levels (LN: 0.45 mM NH₄NO₃, HN: 1.5 mM NH₄NO₃). Leaves were harvested and total protein was extracted using an in vitro degradation rice lysate and incubated with the in vitro proteins. (A) Comparison of in vitro degradation rates of DEP1-His. (B) Comparison of in vitro degradation rates of dep1-1-His. HSP82 was used as an internal control for sample loading.

[0131] Figure 8. (A) Luciferase complementation assay to detect the interaction between DABE1 and DEP1 and dep1-1 proteins. (B) Co-IP assay to analyze the interaction between DABE1 and DEP1 and dep1-1 proteins.

[0132] Figure 9. (A-C) Statistical plots of plant height (A), tiller number (B), and grain number per ear (C) of a DABE1-overexpressing transgenic line (pActin::DABE1) and a CRISPR-cas9 knockout line (dabe1). Values ​​are mean ± SD (n = 15). Different letters indicate significant differences (P < 0.05). Significance analysis was performed using the Duncan test.

[0133] Figure 10. pActin::DABE1 overexpressing transgenic lines and dabe1 knockout lines grown under different nitrogen levels (LN, 120 kg / ha; HN, 300 kg / ha). (A) Plant type comparison. Scale bar, 20 cm. (B) Nitrogen response analysis of plant height. (C) Nitrogen response analysis of tiller number. (D) Nitrogen response analysis of grain number per spike. Values ​​in the figures are mean ± SD (n = 15). Different letters indicate significant differences (P < 0.05). Significance analysis was performed using the Duncan test.

[0134] Figure 11. WYJ7-DEP1, the DEP1 complete deletion mutant WYJ7-DEP1-C1, the DABE1-overexpressing transgenic line WYJ7-dep1-C1pActin::DABE1 in the WYJ7-dep1-C1 background, and the DABE1 knockout line WYJ7-dep1-C-dabe1 in the WYJ7-dep1-C1 background were grown under different nitrogen levels (LN, 120 kg / ha; HN, 300 kg / ha). (A) Plant architecture comparison. Scale bar, 20 cm. (B) Plant height, (C) Tiller number, (D) Grain number per spike. Values ​​are mean ± SD (n = 15). Different letters indicate significant differences (P < 0.05). Significance analysis was performed using the Duncan test.

[0135] Figure 12. WYJ7-DEP1, the DEP1 complete deletion mutant WYJ7-DEP1-C1, the DABE1-overexpressing transgenic line WYJ7-dep1-C1pActin::DABE1 in the WYJ7-dep1-C1 background, and the DABE1 knockout line WYJ7-dep1-C-dabe1 in the WYJ7-dep1-C1 background were grown under different nitrogen levels (LN, 120 kg / ha; HN, 300 kg / ha) and analyzed for nitrogen responses. (A) Nitrogen response analysis of plant height, (B) nitrogen response analysis of tiller number, and (C) nitrogen response analysis of grain number per spike. Values ​​in the figures are mean ± SD (n = 15). Different letters indicate significant differences (P < 0.05). Significance was analyzed using the Duncan test.

[0136] Figure 13. WYJ7-dep1-1, the DABE1-overexpressing transgenic line WYJ7-dep1-1 pActin::DABE1 under the WYJ7-dep1-1 background, and the DABE1-knockout line WYJ7-dep1-1 dabe1 under the WYJ7-dep1-1 background were grown under different nitrogen levels (LN, 120 kg / ha; HN, 300 kg / ha). (A) Plant type, scale bar, 20 cm, (B) Plant height, (C) Tiller number, (D) Grain number per spike. Values ​​are mean ± SD (n = 15). Different letters indicate significant differences (P < 0.05). Significance analysis was performed using the Duncan test.

[0137] Figure 14. WYJ7-dep1-1, the DABE1-overexpressing transgenic line WYJ7-dep1-1 pActin::DABE1 under the WYJ7-dep1-1 background, and the DABE1-knockout line WYJ7-dep1-1 dabe1 under the WYJ7-dep1-1 background were grown under different nitrogen levels (LN, 120 kg / ha; HN, 300 kg / ha) and analyzed for nitrogen responses. (A) Nitrogen response in plant height, (B) nitrogen response in tiller number, and (C) nitrogen response in grain number per spike. Values ​​are mean ± SD (n = 15). Different letters indicate significant differences (P < 0.05). Significance was analyzed using the Duncan test.

[0138] Figure 15. (A) Ubiquitination levels of the DEP1-GFP fusion protein in transgenic rice lines pActin::DEP1-GFP and pActin::DEP1-GFP pActin::DABE1. (B) Grayscale statistics of the relative ubiquitination levels of the DEP1-GFP fusion protein in pActin::DEP1-GFP and pActin::DEP1-GFP pActin::DABE1, analyzed from three independent experiments.

[0139] Figure 16. (A) Analysis of the degradation rate of DEP1-His using an in vitro protein degradation assay. Lysates of WYJ7-DEP1, pActin::DABE1, and dabe1 rice plants were incubated with DEP1-His protein in the presence of MG132. HSP82 was used as an internal control for sample loading. (B) WYJ7-dep1-C1 pActin::DEP1-GFP, pActin::DEP1-GFP WYJ7-dep1-C1-dabe1, and pActin::DEP1-GFP WYJ7-dep1-C1-dabe1 pActin::DABE1 rice plants were cultured under different nitrogen levels (LN: 0.45 mM NH₄NO₃; HN: 1.5 mM NH₄NO₃), and the accumulation of the DEP1-GFP fusion protein was measured. HSP82 was used as an internal control for sample loading.

[0140] Figure 17. Phylogenetic tree analysis of the DABE1 gene in rice and Arabidopsis.

[0141] Figure 18. (A) Analysis of DEP1 ubiquitination using a rice protoplast system. Myc-DEP1 was expressed alone or co-expressed with Flag-DABE1 (Flag-DABE2, Flag-DABE3) in rice protoplasts. (B) Analysis of the degradation rate of DEP1-His using an in vitro protein degradation assay. Rice lysates were incubated with GST-DABE2 or GST-DABE3 and the degradation rate of the DEP1-His fusion protein was analyzed.

[0142] Figure 19. Comparative analysis of yield traits of WYJ7-DEP1, dabe1, and dabe1 dabe2 dabe3 rice accessions. (A) Plant type, scale bar, 20 cm. (B) Panicle type, scale bar, 5 cm. (C) Plant height. (D) Tiller number. (E) Number of grains per panicle. (F) 1000-grain weight. (G) Yield per plant. (G) Yield of plots under different nitrogen levels (60 kg / ha, 120 kg / ha, 210 kg / ha, and 300 kg / ha). Values ​​in the figure are mean ± SD (n = 15). Different letters indicate significant differences (P < 0.05). Significance analysis was performed using the Duncan test.

[0143] Figure 20. Analysis of rice appearance quality traits of WYJ7-DEP1, dabe1, and dabe1 dabe2 dabe3. (A) Grain shape, scale 5 mm. (B) Grain length and width. (C) Grain length-to-width ratio. (D) 1000-grain weight. (E) Scanning electron microscopy image of a cross-section of a starch granule. (F) Grain chalkiness. (G) Grain transparency. Values ​​in the figures are mean ± SD (n = 15). Different letters indicate significant differences (P < 0.05). Significance analysis was performed using the Duncan test. DETAILED DESCRIPTION

[0144] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments and with reference to the accompanying drawings.

[0145] The experimental methods in the following examples are all conventional experimental methods unless otherwise specified. The reagents, kits, and experimental instruments used in the experiments can all be purchased from biological instrument and reagent companies unless otherwise specified.

[0146] Example 1: DEP1 protein accumulation is positively regulated by nitrogen levels

[0147] It has been reported that by crossing Nipponbare (carrying the DEP1 locus) with Wuyunjing 7 (WYJ7-dep1-1), carrying the dep1-1 locus, and backcrossing Wuyunjing 7 multiple times, a pair of near-isogenic lines, WYJ7-DEP1 and WYJ7-dep1-1, were obtained. WYJ7-dep1-1 has a semi-dwarf phenotype, erect and dense panicles, and a partial loss of nitrogen response during vegetative growth (Huang et al., 2009; Sun et al., 2014). This suggests that DEP1 is involved in regulating the nitrogen response of rice growth and development. To genetically prove that DEP1 is involved in regulating the nitrogen response of rice growth, the present inventors designed primers from the rice DEP1 gene to connect the target sequence to the sgRNA expression cassette (sequence SEQ ID NO: 10), and inserted it into the pYLCRISPR / Cas9P ubi-MH, using the method described in the literature (Ma et al., 2015). A complete loss-of-function mutant of DEP1, WYJ7-dep1-C1, was obtained in the WYJ7-dep1-1 background. The inventors cultivated WYJ7-DEP1 and WYJ7-dep1-C1 under different nitrogen application rates. Phenotypic analysis revealed that under high nitrogen levels (HN, 300 kg / ha), WYJ7-dep1-C1 exhibited significantly shorter plant height (Figure 1A), significantly fewer tillers (Figure 1B), and significantly fewer grains per panicle (Figure 1C) compared to WYJ7-DEP1. Compared to high nitrogen levels, under low nitrogen levels (LN, 120 kg / ha), WYJ7-DEP1 exhibited significantly lower plant height, tiller number, and grain number per panicle, while WYJ7-dep1-C1 exhibited no significant changes in plant height, tiller number, and grain number per panicle (Figures 1A, B, C). Furthermore, to characterize rice growth response to nitrogen levels, the inventors introduced the Nitrogen-Mediated Growth Response (NGR) formula (agronomic traits under high nitrogen - agronomic traits under low nitrogen) / agronomic traits under low nitrogen) as a measure of rice agronomic sensitivity to nitrogen. Compared with WYJ7-DEP1, the NGR values ​​of plant height, tiller number, and grain number per panicle in WYJ7-dep1-1 were significantly reduced (Figure 2A, B, C); the NGR values ​​of plant height, tiller number, and grain number per panicle in WYJ7-dep1-C1 were further reduced (Figure 2A, B, C). These results suggest that DEP1 is involved in the nitrogen response process of rice growth.

[0148] The present inventors designed the following primers according to conventional molecular biology and genetic manipulation methods based on the promoter sequence of the DEP1 gene (SEQ ID NO: 12):

[0149] Primers used when constructing the promoter of the DEP1 gene into a vector (pCAMBIA2300, which is commercially available, for example, from Shanghai Lianmai Biotechnology Co., Ltd.) are: pDEP1-F (SEQ ID NO: 14) and pDEP1-R (SEQ ID NO: 15).

[0150] Primers used when constructing the cDNA sequence of DEP1 (SEQ ID NO: 11) or the cDNA sequence of dep1-1 (SEQ ID NO: 13) into the vector pCAMBIA2300: DEP1-F (SEQ ID NO: 16), DEP1-R (SEQ ID NO: 17), dep1-1-F (SEQ ID NO: 18), and dep1-1-R (SEQ ID NO: 19).

[0151] Using DNA from WYJ7-DEP1 and WYJ7-dep1-1 as templates, the pCAMBIA2300 vector backbone was used, along with the Gateway BP Clonase II Enzyme mix and Gateway LR Clonase II Enzyme mix kits (purchased from Invitrogen) to construct the pDEP1::DEP1-GFP and pDEP1::dep1-1-GFP vectors, respectively. The WYJ7-dep1-C1 mutant was transformed using Agrobacterium-mediated transformation, yielding the positive transgenic lines WYJ7-dep1-C1pDEP1::DEP1-GFP and WYJ7-dep1-C1pDEP1::dep1-1-GFP. The positive transgenic lines were grown under high and low nitrogen conditions and the nitrogen response (NGR) of plant height, tiller number, and grain number per ear was analyzed. The results showed that the nitrogen response values ​​(NGR) of plant height, tiller number, and grain number per spike of WYJ7-dep1-C1 pDEP1::DEP1-GFP were comparable to those of WYJ7-DEP1 (Figure 2A, B, C); while the nitrogen response values ​​(NGR) of plant height, tiller number, and grain number per spike of WYJ7-dep1-C1 pDEP1::dep1-1-GFP were comparable to those of WYJ7-dep1-1 (Figure 2A, B, C).

[0152] The above results show that DEP1 is a key gene regulating the nitrogen response of rice growth and development; and the dep1-1 gene can reduce the response of rice growth to nitrogen.

[0153] To investigate how DEP1 regulates rice growth and development in response to nitrogen levels, the inventors planted rice plants in the field at different nitrogen application rates (60 kg / ha, 120 kg / ha, 210 kg / ha, and 300 kg / ha). Leaf RNA was extracted and quantitative PCR was used to analyze DEP1 or dep1-1 transcript levels. The authors found that the transcript levels of both DEP1-GFP and dep1-1-GFP increased with increasing nitrogen levels (Figure 3A). Furthermore, total protein was extracted from leaves and the accumulation of DEP1-GFP or dep1-1-GFP fusion proteins was analyzed by Western blot using an anti-GFP antibody. For detailed procedures, see (Wu et al., 2020). The results showed that DEP1-GFP protein accumulation was low under low nitrogen levels and increased with increasing nitrogen levels. However, the accumulation of the dep1-1-GFP fusion protein was higher than that of DEP1-GFP under both high and low nitrogen conditions, and the accumulation of the dep1-1-GFP fusion protein was insensitive to nitrogen levels (Figure 3B). Therefore, the stability of DEP1 protein increases with the increase of nitrogen level; dep1-1 protein is relatively stable under different nitrogen levels, and its protein stability is not sensitive to different nitrogen levels.

[0154] Example 2: Degradation of DEP1 protein depends on the 26S proteasome

[0155] The present inventors designed the following primers according to conventional molecular biology and genetic manipulation methods based on the cDNA sequences of DEP1 and dep1-1 obtained in Example 1 (SEQ ID NO: 13): DEP1-F (SEQ ID NO: 16), DEP1-R (SEQ ID NO: 17), dep1-1-F (SEQ ID NO: 18) and dep1-1-R (SEQ ID NO: 19).

[0156] The inventors extracted total RNA from leaves of a pair of rice materials, WYJ7-DEP1 and WYJ7-dep1-1, and reverse transcribed the cDNA to synthesize cDNA. The cDNA was used as a template to amplify the CDS of DEP1 and dep1-1 using primers DEP1-F and DEP1-R, dep1-1-F and dep1-1-R, respectively. The CDSs were then ligated into the pCAMBIA2300 vector backbone using Gateway BP Clonase II Enzyme mix and Gateway LR Clonase II Enzyme mix kits (purchased from Invitrogen) to construct pActin::DEP1-GFP and pActin::dep1-1-GFP vectors, respectively. The specific operation method can be found in (Wu et al. al., 2020). Furthermore, the WYJ7-dep1-C1 mutant was transformed using Agrobacterium-mediated transfection to obtain positive transgenic plants (Figure 4A). These transgenic plants were then planted under different nitrogen fertilizer conditions (LN, 120 kg / ha; HN, 300 kg / ha). First, phenotypic analysis showed that compared with the WYJ7-dep1-C1 mutant, the pActin::DEP1-GFP transgenic lines showed significantly increased responses to nitrogen levels in terms of plant height, tiller number, and grain number per ear, i.e., NGR values, reaching levels comparable to those of WYJ7-DEP1 (Figure 4B, C, D). However, the pActin::dep1-1-GFP transgenic lines showed a weakened response to nitrogen levels in terms of plant height, tiller number, and grain number per ear, as shown by insignificant increases in NGR values ​​compared to WYJ7-dep1-C1 (Figure 4B, C, D).

[0157] The present inventors then extracted total RNA from the leaves of the above-mentioned materials and reverse-transcribed it into cDNA. The DEP1-GFP / dep1-1-GFP quantitative PCR primers were designed as follows: GFP-F (SEQ ID NO: 20), GFP-R (SEQ ID NO: 21). The transcription levels of DEP1-GFP / dep1-1-GFP were analyzed by fluorescence quantitative PCR, following the procedures described in (Wu et al., 2020). The results showed that under different nitrogen application levels (LN, 120 kg / ha; HN, 300 kg / ha), the transcription levels of DEP1-GFP and dep1-1-GFP in transgenic plants were comparable and unaffected by nitrogen levels (Figure 4A). Total protein from the leaves was further extracted and analyzed by western blot. The results showed that DEP1-GFP protein levels were higher under high nitrogen application and lower under low nitrogen application. However, dep1-1-GFP protein levels remained largely unchanged across different nitrogen levels, and at the same nitrogen level, dep1-1-GFP protein levels were higher than DEP1-GFP protein levels (Figure 4B).

[0158] The inventors cultivated pActin::DEP1-GFP and pActin::dep1-1-GFP transgenic lines under high- and low-nitrogen hydroponic conditions (HN, 1.5mM NH4NO3; LN, 0.45mM NH4NO3), respectively, and treated them with 50μM MG132 for 6 hours. Total protein was extracted from the leaves, and protein levels were analyzed by western blot. The results showed that MG132 treatment increased DEP1-GFP protein levels under both high and low nitrogen conditions. DEP1-GFP protein levels were not affected by nitrogen levels and remained high under low nitrogen conditions. However, MG132 treatment did not affect dep1-1-GFP protein levels under both high and low nitrogen conditions (Figure 6A). Accumulation of the dep1-1-GFP fusion protein was high before and after MG132 treatment (Figure 6A).

[0159] The present inventors used conventional molecular biology methods to extract total protein from the leaves of the pActin::DEP1-GFP transgenic line, enriched the DEP1-GFP protein using GFP beads, and performed western-blot analysis. Anti-Ub ubiquitin antibody (purchased from Abcam) was used to detect that the DEP1-GFP protein was ubiquitinated ( Figure 6B ). For specific procedures, see ( Wu et al., 2020 ).

[0160] To detect the difference in the stability of DEP1 and dep1-1 proteins, the Pichia pastoris eukaryotic expression system (Invitrogen, EasySelect TMPastoris expression kit) was used to express and purify DEP1-His and dep1-1-His proteins (for detailed operations, see the kit instructions). The inventors used in vitro protein degradation buffer (25mM Tris-HCl, pH 7.5; 10mM NaCl; 10mM MgCl2; 4mM PMSF; 5mM dithiothreitol; 10mM ATP) to extract total protein from WYJ7-DEP1 rice leaves grown for 4 weeks under high and low nitrogen hydroponic conditions (HN, 1.5mM NH4NO3; LN, 0.45mM NH4NO3). Equal amounts of the above-mentioned eukaryotically expressed DEP1-His and dep1-1-His fusion proteins were added, and then 50μM MG132 was added. The reaction was terminated at 0, 3, 9, and 18 minutes, respectively. Changes in the levels of DEP1-His and dep1-1-His fusion proteins were detected by western blot. The results showed that the DEP1-His protein stability in rice lysate under high nitrogen conditions was higher than that in rice lysate under low nitrogen conditions (Figure 7A); the dep1-1-His fusion protein was highly stable under both high and low nitrogen conditions, and its fusion protein stability was insensitive to changes in nitrogen levels (Figure 7B); after MG132 treatment, the protein stability of DEP1-His under low nitrogen conditions returned to the protein stability under high nitrogen conditions, and its protein stability also reduced its sensitivity to changes in nitrogen levels.

[0161] The above results show that DEP1 protein stability is high under high nitrogen conditions, while DEP1 protein stability is low under low nitrogen conditions, and the regulation of DEP1 protein stability by nitrogen levels depends on the 26s proteasome; dep1-1 protein stability is higher than DEP1 under low nitrogen conditions, and the protein stability of dep1-1 is not sensitive to different nitrogen levels.

[0162] Example 3: Identification and functional analysis of DEP1-interacting protein DABE1

[0163] The present inventors constructed DEP1 into the yeast two-hybrid vector pGBKT7 (commercially available, for example, from U-Bio) by conventional molecular biology methods. The primer sequences used to construct the vector are as follows: Do-DEP1-F1 (SEQ ID NO: 22), Do-DEP1-R1 (SEQ ID NO: 22).

[0164] Using DEP1 as bait protein and GAL4 as a reporter gene, a yeast two-hybrid system was used to screen a yeast library constructed from rice cDNA. The DEP1-interacting protein, DABE1, was identified. It contains a BTB domain. Previous studies have shown that BTB domain-containing proteins often form complexes with cullin3 to promote substrate ubiquitination, thereby mediating the substrate's degradation by the 26S proteasome.

[0165] The present inventors designed the following primers according to conventional molecular biological procedures based on the cDNA sequences of DEP1 and dep1-1 and the cDNA sequence of the DABE1 gene (SEQ ID NO: 1): Do-DEP1-F1 (SEQ ID NO: 22), Do-DEP1-R1 (SEQ ID NO: 23), Do-dep1-1-F1 (SEQ ID NO: 24), Do-dep1-1-R1 (SEQ ID NO: 25), Do-DABE1-F (SEQ ID NO: 26) and Do-DABE1-R (SEQ ID NO: 27).

[0166] Using the pCAMBIA2300 vector backbone, nLUC-DEP1, nLUC-dep1-1, and cLUC-DABE1 plasmids were constructed and transformed into Agrobacterium. Single colonies were picked and shaken, then suspended in resuspension buffer (150 μM acetosyringone, 10 mM MgCl2, 10 mM MES-KOH pH 5.7) to adjust the OD of the bacterial solution of nLUC and cLUC vectors. 600 =1.0, while adjusting the OD of Agrobacterium overexpressing tomato dwarf virus p19 (to prevent exogenous gene silencing) 600 = 0.5 (Voinnet et al., 2003). After 3 hours of stabilization, nLUC, cLUC, and p19 were mixed at a ratio of 5:5:2 and injected into the lower epidermis of 3-week-old tobacco plants using a 1 mL syringe. Two to three days later, protein interactions were observed using laser confocal microscopy. The results showed that DABE1 interacted with DEP1 but not with dep1-1 (Figure 8A).

[0167] In addition, the present inventors designed the following primers according to conventional molecular biological operation methods based on the cDNA sequences of DEP1 and dep1-1 and the cDNA sequence of the DABE1 gene (SEQ ID NO: 1): Do-DEP1-F1 (SEQ ID NO: 22), Do-DEP1-R1 (SEQ ID NO: 23), Do-dep1-1-F1 (SEQ ID NO: 24), Do-dep1-1-R1 (SEQ ID NO: 25), Do-DABE1-F (SEQ ID NO: 26) and Do-DABE1-R (SEQ ID NO: 27).

[0168] Using the PUC19 vector backbone (purchased from Takara), DABE1-Flag, DEP1-HA, and dep1-1-HA vectors were constructed and transformed into rice protoplasts. Rice protoplast co-immunoprecipitation (Co-IP) is an experimental method for studying protein interactions in vivo. Protoplasts were prepared by dissociating leaf sheaths from sterile WYJ-DEP1 rice seedlings cultured for approximately 10 days. Transformations were performed overnight with DABE1-Flag as a control group and DEP1-HA, DABE1-Flag, dep1-1-HA, and DABE1-Flag as experimental groups. Total protoplast protein was extracted, and 60 μL of total protein was mixed with an appropriate amount of SDS loading buffer and boiled for 10 minutes. The remaining total protein supernatant from the experimental and control groups was incubated with anti-Flag antibody-encapsulated magnetic beads at 4°C for 1 hour. The beads were then rinsed 3-4 times with protein wash buffer, the supernatant discarded, and the beads were mixed with an appropriate amount of SDS loading buffer and boiled for 10 minutes. Protein interactions were then analyzed by SDS-PAGE electrophoresis and Western blot. The results showed that DABE1 interacted with DEP1 but not with dep1-1 (Figure 8B). These results indicate that DABE1 protein can interact with DEP1 protein but not with dep1-1 protein, and that the cysteine-rich region at the C-terminus of DEP1 protein may be the key domain for the interaction between DEP1 and DABE1.

[0169] Example 4: DABE1 is a negative regulator of rice grain number

[0170] To further study the function of DABE1, the present inventors designed the following primers based on the DABE1 gene cDNA sequence (SEQ ID NO: 1) using conventional molecular biology and genetic manipulation methods:

[0171] Do-DABE1-F (SEQ ID NO: 26) and Do-DABE1-R (SEQ ID NO: 27) were amplified by PCR using primers DABE1-F and DABE1-R. The fragments were ligated into the pCAMBIA2300 vector backbone to construct the pActin::DABE1 vector, which was then transformed into WYJ7-DEP1 using Agrobacterium-mediated transformation to obtain positive transgenic plants.

[0172] The present inventors designed primers based on the rice DABE1 gene, connected the target sequence into the sgRNA expression cassette (sequence SEQ ID NO: 7), and inserted it into the pYLCRISPR / Cas9P ubi -MH was used, following the method described in the literature (Ma et al., 2015). WYJ7-DEP1 was transformed using Agrobacterium-mediated transfection to construct the dabe1 knockout line. WYJ7-DEP1, pActin::DABE1, and dabe1 were grown under high and low nitrogen conditions (LN, 120 kg / ha; HN, 300 kg / ha). Phenotypic analysis showed that compared with WYJ7-DEP1, pActin::DABE1 transgenic plants exhibited shorter plant height (Figure 9A), fewer tillers (Figure 9B), and significantly fewer grains per panicle (Figure 9C). Compared with wild-type WYJ7-DEP1, the dabe1 mutant exhibited slightly shorter plant height (Figure 9A), no significant difference in tiller number (Figure 9B), and significantly increased grain number per panicle (Figure 9C). These results suggest that DABE1 inhibits rice growth and development and has a significant negative regulatory effect on grain number per panicle.

[0173] Example 5: DABE1-mediated regulation of rice grain number in response to nitrogen

[0174] The present inventors planted WYJ7-DEP1, pActin::DABE1, and dabe1 in fields under different nitrogen application levels (LN, 120 kg / ha; HN, 300 kg / ha) and conducted phenotypic analysis. The results showed that WYJ7-DEP1 exhibited a normal nitrogen response under different nitrogen application levels, with reduced plant height, fewer grains per spike, and fewer tillers under low nitrogen conditions (Figures 9A-C). However, pActin::DABE1 showed no significant differences in plant height, tiller number, or grain number per spike across different nitrogen application levels (Figures 9A-C). Similarly, dabe1 also showed no significant differences in plant height, tiller number, or grain number per spike across different nitrogen application levels (Figures 9A-C). In addition, the NGR analysis results of the responses of plant height, tiller number, and grain number per ear to external nitrogen application levels showed that compared with WYJ7-DEP1, the responses of pActin::DABE1 and dabe1 to different nitrogen application levels in terms of plant height, tiller number, and grain number per ear were weakened (Figure 10B-D).

[0175] The above results indicate that DABE1 is involved in regulating the nitrogen response of grain number per spike, and it is speculated that DABE1 and DEP1 are in the same genetic regulatory network in regulating the nitrogen response of grain number per spike.

[0176] Example 6: DABE1-DEP1 molecular module regulates nitrogen response of rice grain number per panicle

[0177] The present inventors hybridized the pActin::DABE1 and dabe1 in Example 6 with the DEP1 complete deletion mutant WYJ7-DEP1-C1 in Example 1, respectively, and obtained the pActin::DABE1 homozygous strain and the DABE1 knockout strain dabe1 in the WYJ7-DEP1-C1 background, respectively. They were planted under high and low nitrogen planting conditions (LN, 120 kg / ha; HN, 300 kg / ha) and the phenotypes were analyzed. The results showed that WYJ7-DEP1 exhibited normal nitrogen responses under both high and low nitrogen levels. For example, under low nitrogen, its plant height decreased (Figure 11B), tiller number decreased (Figure 11C), and grain number per panicle decreased (Figure 11D). In contrast, the pActin::DABE1 and dabe1 knockout lines in the WYJ7-DEP1-C1 background showed similar plant height, tiller number, and grain number per panicle to those of WYJ7-DEP1-C1 under both high and low nitrogen conditions, with significantly lower values ​​than those of WYJ7-DEP1 (Figures 11B-D). Furthermore, the nitrogen response (NGR) values ​​for plant height, tiller number, and grain number per panicle were not significantly different from those of WYJ7-DEP1-C1 (Figures 12A-C). These results suggest that DABE1 is dependent on DEP1 in regulating the nitrogen response of rice grain number per panicle and is genetically upstream of DEP1.

[0178] To further explore the genetic interaction between DABE1 and dep1-1, the present inventors hybridized the overexpressing transgenic material pActin::DABE1 and the knockout material dabe1 in the WYJ7-DEP1 background into the DEP1 gain-of-function mutant WYJ7-dep1-1 background, and backcrossed WYJ7-dep1-1 twice to obtain DABE1 overexpressing transgenic lines and dabe1 knockout materials in the WYJ7-dep1-1 background ( Figure 13A ). The plant height, tiller number, and grain number per ear of the DABE1 overexpressing transgenic lines and knockout lines in the WYJ7-dep1-1 background were not significantly different from those of WYJ7-dep1-1 ( Figures 13B-D ), and their NGR values ​​for plant height, tiller number, and grain number per ear were also not significantly different from those of WYJ7-dep1-1 ( Figures 14A-C ). The above results indicate that dep1-1 regulates rice grain number per panicle and nitrogen response of grain number per panicle independently of DABE1.

[0179] Example 7: DABE1 mediates ubiquitination of DEP1 protein

[0180] The present inventors hybridized the pActin::DEP1-GFP transgenic line in the WYJ7-dep1-C1 background of Example 2 with the pActin::DABE1 transgenic line of Example 6 to obtain the homozygous line pActin::DEP1-GFP pActin::DABE1, which co-expresses DEP1 and DABE1. Total protein from seedlings of these lines was extracted and ubiquitinated proteins in vivo were immunoprecipitated using p62-derived UBA domain agarose beads. Anti-GFP antibodies were then used for detection. The results showed that compared to pActin::DEP1-GFP, the ubiquitination level of DEP1-GFP protein in pActin::DEP1-GFP pActin::DABE1 was significantly higher ( FIG15A ). This conclusion was further confirmed by grayscale value statistics of the relative ubiquitination level of DEP1 in three independent experiments ( FIG15B ). This experiment demonstrated that DABE1 can promote DEP1 ubiquitination. In other words, DEP1 is a substrate for DABE1 as an E3 ubiquitin ligase.

[0181] Example 8: Low nitrogen-induced DEP1 protein degradation is dependent on DABE1

[0182] The inventors demonstrated that DABE1 affects the stability of DEP1 protein through in vitro degradation experiments. The specific method is as follows: using in vitro protein degradation buffer (25mM Tris-HCl, pH 7.5, 10mM NaCl, 10mM MgCl2, 4mM PMSF, 5mM dithiothreitol, 10mM ATP) to extract total protein from leaves of WYJ7-DEP1, the pActin::DABE1 overexpression strain in Example 5, and the dabe1 knockout strain grown in the field for 4 weeks as a reaction system. The above extracts were added to the DEP1-His fusion protein, and then 50μM MG132 was added. The reaction was terminated at 0, 3, 9, and 18 minutes, respectively, and the changes in the DEP1-His fusion protein were detected by western blot. The results showed that compared with WYJ7-DEP1, the degradation rate of DEP1-His fusion protein in DABE1 was significantly slower, while that in pActin::DABE1 was significantly faster ( FIG16A ), indicating that DABE1 negatively regulates the stability of DEP1 protein.

[0183] On this basis, the inventors hybridized the pActin::DEP1-GFP of Example 3 with the pActin::DABE1 and dabe1 in Example 5, respectively, to obtain overexpression and knockout strains of DABE1 in the homozygous pActin::DEP1-GFP background. Western blot was used to detect the accumulation levels of DEP1-GFP fusion protein in these materials under high and low nitrogen levels (HN, 1.5mM NH4NO3; LN, 0.45mM NH4NO3). The results showed that the accumulation level of DEP1-GFP fusion protein in the control pActin::DEP1-GFP was higher under high nitrogen conditions, while the accumulation level was lower under low nitrogen conditions; in comparison, the accumulation level of DEP1-GFP fusion protein in DABE1 was not affected by nitrogen levels and was higher under both high and low nitrogen conditions; the accumulation level of DEP1-GFP fusion protein in the DABE1 overexpression strain was also not affected by nitrogen levels and was lower under both high and low nitrogen conditions (Figure 16B). The above results indicate that DABE1 negatively regulates DEP1 protein stability and that DABE1 mediates nitrogen regulation of DEP1 stability.

[0184] Example 9: DABE1 homologous proteins can promote DEP1 protein degradation

[0185] The present inventors used the PLAZA website to perform sequence analysis of DABE1 homologous proteins and used MEGA to construct a phylogenetic tree of DABE1 in rice and Arabidopsis ( FIG17 ), and found that DABE1 has two homologous genes: DABE2 (LOC_Os12g40480) and DABE3 (LOC_Os07g03150).

[0186] The present inventors designed the following primers based on the cDNA sequences of DABE1, DABE2, and DABE3 genes according to conventional molecular biological procedures: Do-DABE1-F (SEQ ID NO: 26) and Do-DABE1-R (SEQ ID NO: 27), Do-DABE2-F (SEQ ID NO: 28) and Do-DABE2-R (SEQ ID NO: 29), Do-DABE3-F (SEQ ID NO: 30) and Do-DABE3-R (SEQ ID NO: 31), and Do-Ub-F (SEQ ID NO: 32) and Do-Ub-R (SEQ ID NO: 33).

[0187] Using these primers, the target fragment was amplified using genomic DNA from the WYJ7-DEP1 wild-type rice strain as a template and ligated into the PUC19 vector backbone to construct DABE1-Flag, DABE2-Flag, DABE3-Flag, Myc-DEP1, and Ub-HA vectors, which were then transformed into rice protoplasts. Total protoplast protein was extracted, and after immunoprecipitation of the DEP1 protein using anti-Myc magnetic beads (purchased from Abcam), the ubiquitination level of the Myc-DEP1 fusion protein was detected using an anti-HA antibody. The results showed that co-expression of Myc-DEP1 with DABE1-Flag, DABE2-Flag, or DABE3-Flag increased the ubiquitination level of the Myc-DEP1 fusion protein compared to expression of Myc-DEP1 alone (Figure 18A). These results indicate that DABE1, DABE2, and DABE3 can all increase the ubiquitination level of the DEP1 protein.

[0188] The present inventors verified whether DABE2 and DABE3 affect the stability of DEP1 protein through in vitro degradation experiments. The specific method is as follows: Total protein from WYJ7-DEP1 wild-type leaves grown in the field for 4 weeks was extracted using in vitro protein degradation buffer (25mM Tris-HCl, pH 7.5; 10mM NaCl; 10mM MgCl2; 4mM PMSF; 5mM dithiothreitol; 10mM ATP) as the reaction system. The above extract was divided and added to the DEP1-His fusion protein in Example 2, and then GST-DABE2 or GST-DABE3 protein expressed using a prokaryotic expression system and purified using GST agarose beads was added. The reaction was terminated at 0, 3, 9, and 18 minutes, respectively, and the level of DEP1-His fusion protein was detected by western blot. The results showed that the additional addition of GST-DABE2 or GST-DABE3 to the experimental system significantly accelerated the degradation rate of DEP1-His protein (Figure 18B). These results suggest that, similar to DABE1, the homologous proteins DABE2 and DABE3 can promote the degradation of DEP1 by increasing the ubiquitination level of the DEP1 protein. Therefore, DABE1 and its homologous genes DABE2 and DABE3 have similar functions, indicating that the three are functionally conserved.

[0189] Example 10: Mutations in DABE1 and its homologous genes can increase rice yield and nitrogen fertilizer use efficiency

[0190] Given that DABE1 and its homologous genes DABE2 and DABE2 are functionally conserved, the present inventors further created three knockout strains of DABE1 (SEQ ID NO: 1), DABE2 (SEQ ID NO: 3), and DABE3 (SEQ ID NO: 5) based on the knockout strain dabe1 created in Example 4 to identify and compare the effects of single gene knockout of DABE1 and triple gene knockout of DABE1, DABE2, and DABE3 on rice nitrogen fertilizer use efficiency, yield, and rice quality. The specific method is: based on the sequence homology of DABE1, DABE2, and DABE2, primers are designed and PCR amplified and the target sequence is connected to the sgRNA expression cassette, as shown in (sequence SEQ ID NO: 7, SEQ ID NO: 8, sequence SEQ ID NO: 9), and the enzyme digestion and ligation are inserted into the pYLCRISPR / Cas9P ubi-MH, using the method described in the literature (Ma et al., 2015), was transformed into WYJ7-DEP1 using Agrobacterium-mediated transformation, and the DABE1, DABE2, and DABE3 triple knockout strains (dabe1 dabe2 dabe3) were constructed in the WYJ7-DEP1 background. WYJ7-DEP1, dabe1, and dabe1 dabe2 dabe3 were planted in the field under different nitrogen fertilizer levels and their phenotypes were analyzed. The results showed that under normal nitrogen application levels, the plant height of dabe1 and dabe1 dabe2 dabe3 was slightly reduced compared with WYJ7-DEP1 (Figure 19C); compared with WYJ7-DEP1, the tiller number of dabe1 did not change significantly, while the tiller number of dabe1 dabe2 dabe3 decreased by about 20% (Figure 19D); compared with WYJ7-DEP1, the grain number per spike of dabe1 increased by about 30%, while the grain number per spike of dabe1 dabe2 dabe3 increased by about 40% (Figures 19B,E); compared with WYJ7-DEP1, the yield per plant of dabe1 increased by about 8%, while the yield per plant of dabe1 dabe2 dabe3 increased by about 16% (Figure 19F). Compared to WYJ7-DEP1, both dabe1 and dabe1 dabe2 dabe3 achieved higher plot yields at lower nitrogen levels, with dabe1 dabe2 dabe3 exhibiting a yield-enhancing effect superior to dabe1. At the lowest field nitrogen level (60 kg / ha), dabe1 dabe2 dabe3 achieved a yield increase of approximately 40% (Figure 19G). Furthermore, at a moderately reduced nitrogen fertilizer application rate (210 kg / ha), the plot yields of dabe1 dabe2 dabe3 were comparable to those of WYJ7-DEP1 at excess nitrogen fertilizer (300 kg / ha) (Figure 19G), demonstrating improved nitrogen fertilizer use efficiency in rice. These results indicate that DABE1 and its homologous genes DABE2 and DABE3 are important new genes regulating nitrogen response in rice growth and development. Multi-gene knockout (preferably triple knockout of DABE1, DABE2, and DABE3) has a more significant yield-increasing effect under low-nitrogen conditions than single-gene knockout (preferably DABE1). Therefore, DABE1 and its homologous genes DABE2 and DABE3 have important application value in molecular breeding for high-yield and nitrogen-efficient rice.

[0191] Example 11: Loss-of-function mutations in DABE1 and its homologous genes can improve rice appearance quality

[0192] The present inventors analyzed the rice grain length, grain width, aspect ratio, and 1000-grain weight of WYJ7-DEP1, dabe1, and dabe1 dabe2 dabe3. The results showed that compared with WYJ7-DEP1, dabe1 and dabe1 dabe2 dabe3 showed significantly increased grain length but unchanged grain width (Figure 20B), resulting in an improved aspect ratio (Figure 20C) and 1000-grain weight (Figure 20D). Furthermore, scanning electron microscopy images revealed that compared with the wild-type WYJ7-DEP1, the starch granules of dabe1 dabe2 dabe3 were more densely packed, while dabe1 showed no significant differences compared to the wild-type. This suggests that dabe1 dabe2 dabe3 improves rice quality, while dabe1 does not alter rice quality (Figure 20E). Furthermore, compared with WYJ7-DEP1, the chalkiness of dabe1, dabe2, and dabe3 rice grains was significantly reduced (Figure 20F) and their transparency was improved (Figure 20G). However, the chalkiness and transparency of dabe1 rice grains remained unchanged (Figure 20F, G). These results suggest that DABE1, DABE2, and DABE3 have redundant functions in regulating rice appearance quality and need to work together to regulate both chalkiness and transparency. Dabe1, dabe2, and dabe3 can significantly improve multiple aspects of rice appearance quality.

[0193] In general, DABE1 and its homologous genes DABE2 and DABE3 are conserved and functionally redundant in regulating rice nitrogen use efficiency, yield and rice quality. Single gene knockout of DABE1 can improve rice nitrogen use efficiency, yield and rice appearance quality, while triple gene knockout of DABE1, DABE2 and DABE3 can more significantly improve rice nitrogen use efficiency, yield and rice appearance quality.

[0194] It should be noted that the embodiments described above are only used to explain the present invention and do not constitute any limitation of the present invention. The present invention has been described with reference to typical embodiments, but it should be understood that the words used therein are descriptive and explanatory words, rather than restrictive words. The present invention may be modified as specified within the scope of the claims of the present invention, and the present invention may be revised without departing from the scope and spirit of the present invention. Although the present invention described therein relates to specific methods, materials and embodiments, it does not mean that the present invention is limited to the specific examples disclosed therein. On the contrary, the present invention can be extended to all other methods and applications with the same function.

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[0207] Sequence Listing

[0208] SEQ ID NO: 1: Nucleotide sequence of DABE1 gene

[0209] SEQ ID NO: 2: Amino acid sequence of DABE1 protein

[0210] SEQ ID NO: 3: Nucleotide sequence of DABE2 gene

[0211] SEQ ID NO: 4: Amino acid sequence of DABE2 protein

[0212] SEQ ID NO: 5: Nucleotide sequence of DABE3 gene

[0213] SEQ ID NO: 6: Amino acid sequence of DABE3 protein

[0214] SEQ ID NO: 7:

[0215] U6a-sgRNA cassette for monocots Note: The underlined part is the target sequence, and the bold part is the sgRNA coding sequence.

[0216] SEQ ID NO:8:

[0217] U6b-sgRNA cassette for monocots Note: The underlined part is the target sequence, and the bold part is the sgRNA coding sequence.

[0218] SEQ ID NO:9:

[0219] U6c-sgRNA cassette for monocots Note: The underlined part is the target sequence, which is the reverse complementary sequence of the DABE3 gene nucleotide sequence GTGTCATGCATTGAGGTAT, and the bold part is the sgRNA coding sequence

[0220] SEQ ID NO: 10:

[0221] U6a-sgRNA cassette for monocots Note: The underlined part is the target sequence, and the bold part is the sgRNA coding sequence.

[0222] SEQ ID NO:11DEP1 cDNA sequence

[0223] SEQ ID NO: 12DEP1 promoter sequence

[0224] SEQ ID NO:13dep1-1 cDNA sequence

[0225] SEQ ID NO:14

[0226] SEQ ID NO:15

[0227] SEQ ID NO:16

[0228] SEQ ID NO: 17

[0229] SEQ ID NO: 18

[0230] SEQ ID NO: 19

[0231] SEQ ID NO:20

[0232] SEQ ID NO:21

[0233] SEQ ID NO:22

[0234] SEQ ID NO:23

[0235] SEQ ID NO:24

[0236] SEQ ID NO:25

[0237] SEQ ID NO:26

[0238] SEQ ID NO:27

[0239] SEQ ID NO:28

[0240] SEQ ID NO:29

[0241] SEQ ID NO:30

[0242] SEQ ID NO:31

[0243] SEQ ID NO:32

[0244] SEQ ID NO:33

Claims

1. A protein that regulates nitrogen fertilizer utilization, yield and / or grain appearance quality of crops, wherein the amino acid sequence is shown in any one of the following: 1) the amino acid sequence shown in SEQ ID NO: 2, 4, 6; 2) An amino acid sequence that is different from the amino acid sequence shown in SEQ ID NO: 2, 4, 6 due to substitution, deletion and / or insertion of one or more amino acid residues, but has the same activity as the protein composed of the amino acid sequence shown in SEQ ID NO: 2, 4, 6; 3) an amino acid sequence that has at least 70%, preferably at least 80%, and more preferably at least 90% identity to the amino acid sequence shown in SEQ ID NO: 2, 4, or 6, and has the same activity as the protein consisting of the amino acid sequence shown in SEQ ID NO: 2, 4, or 6; 4) An active fragment comprising any one of the amino acid sequences described in 1) to 3).

2. A gene encoding the protein of claim 1, preferably DABE1 or a homologous gene thereof (e.g., DABE2 or DABE3), wherein the nucleotide sequence is as shown in any one of the following: 1) The nucleotide sequences shown in SEQ ID NO: 1, 3, 5; 2) A nucleotide sequence that is different from the nucleotide sequence shown in SEQ ID NO: 1, 3, 5 due to substitution, deletion and / or insertion of one or more nucleotide sequences, but the activity of the protein encoded by it is the same as the activity of the protein encoded by the nucleotide sequence shown in SEQ ID NO: 1, 3, 5; 3) a nucleotide sequence that is at least 70%, preferably at least 80%, and more preferably at least 90% identical to the nucleotide sequence shown in SEQ ID NO: 1, 3, or 5, and the activity of the protein encoded by the nucleotide sequence is the same as that of the protein encoded by the nucleotide sequence shown in SEQ ID NO: 1; 4) Nucleotide sequences that differ in sequence from SEQ ID NO: 1, 3, 5 due to the degeneracy of the genetic code; 5) An active fragment comprising any one of the nucleotide sequences in 1)-4); 6) comprising a nucleotide sequence that hybridizes to a complementary sequence of any one of the nucleotide sequences in 1) to 5) under moderately stringent conditions, preferably highly stringent hybridization conditions; 7) comprising a nucleotide sequence complementary to any one of the nucleotide sequences in 1) to 5).

3. Use of a gene knockout vector or a gene knockout expression cassette of the gene according to claim 2 in cultivating crops with high nitrogen fertilizer utilization efficiency, high yield and / or excellent grain appearance quality, wherein: The crop is a monocotyledonous plant, preferably rice or wheat, more preferably rice.

4. The use according to claim 3, wherein: The gene knockout expression cassette is a sgRNA expression cassette, and the sequence of the sgRNA expression cassette is shown in SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:

9.

5. A method for cultivating crops with high nitrogen fertilizer utilization efficiency, high yield and / or excellent grain appearance quality, the method comprising: The function of the gene described in claim 2 is deleted, thereby obtaining a crop with high nitrogen fertilizer utilization efficiency, high yield and / or excellent grain appearance quality relative to the wild type. Preferably, the function of the gene encoding the control of nitrogen fertilizer utilization efficiency, yield and / or grain appearance quality described in claim 2 is lost by the CRISPR-cas9 gene editing method, and a plant with a single gene knockout of DABE1 or its homologous gene or a plant with multiple gene knockouts of DABE1 and its homologous genes is obtained, thereby obtaining a crop with high nitrogen fertilizer utilization efficiency, high yield and / or excellent grain appearance quality relative to the wild type, wherein the crop is a monocotyledonous plant, preferably rice or wheat, more preferably rice.

6. A method for cultivating crops with high nitrogen fertilizer utilization efficiency, high yield and / or excellent grain appearance quality, the method comprising: A crop plant containing a functional loss mutation of the gene according to claim 2 is hybridized with another plant of the crop to obtain a crop having high nitrogen fertilizer utilization efficiency, high yield and / or excellent grain appearance quality relative to the wild type, wherein the crop is a monocotyledonous plant, preferably rice or wheat, and more preferably rice.

7. The method according to claim 6, wherein the crop plant containing the loss-of-function mutation of the gene according to claim 2 is obtained by gene editing technology, such as CRISPR-cas9.

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