Application of rice osTMK1 protein or its truncated body, encoding gene in regulating plant ear grain number and nitrogen use efficiency

Abstract

The application discloses a rice OsTMK1 protein or a truncated body thereof and a coding gene in regulation of plant ear grain number and nitrogen utilization efficiency. OsTMK1 The gene can increase the ear grain number of rice after overexpression or function enhancement, and can increase the ear grain number of rice under different nitrogen levels, especially under low nitrogen conditions, so that high yield of rice and high efficient utilization of nitrogen are realized. OsTMK1 The gene has important application value for high yield and high efficient nitrogen breeding of crops.

Description

Technical Field

[0001] This invention relates to the fields of plant genetic engineering and plant breeding technology, and more specifically, to the application of rice OsTMK1 protein or its truncated form and encoding gene in regulating the number of grains per panicle and nitrogen fertilizer use efficiency in plants. Background Technology

[0002] The number of grains per panicle is one of the three key factors determining rice yield. Rice grain number is a complex agronomic trait regulated by multiple genes and environmental factors. The total number of grains per panicle is influenced by the number of primary branches, the number of secondary branches, and panicle length. In terms of genetic regulation, several genes regulating rice grain number have been identified through mutagenesis screening and map-based cloning. These genes mainly participate in regulating meristem size and developmental stages, as well as branch development and panicle type. Furthermore, research indicates that plant hormone signaling pathways (auxins, cytokinins, gibberellins, and brassinolide, etc.) interact crisscross and play a crucial role in regulating rice grain number.

[0003] Nitrogen fertilizer is an essential macronutrient for promoting high yields in rice. Studies have found that in both field planting and laboratory hydroponic experiments, the number of primary and secondary branches per panicle in rice increases with increasing nitrogen fertilizer application, leading to a corresponding increase in the total number of grains per panicle. However, the semi-dwarf rice varieties of the "Green Revolution" show reduced nitrogen fertilizer absorption capacity and are not particularly sensitive to nitrogen fertilizer. This results in a diminishing yield increase despite continuously increasing nitrogen fertilizer application, leading to significant nitrogen loss from farmland, increased agricultural production costs, and environmental problems. Therefore, improving crop nitrogen fertilizer utilization efficiency and achieving yield increase while reducing fertilizer use has become a critical issue that urgently needs to be addressed.

[0004] Research reports on high-yield and nitrogen-efficient genes. DEP1 ( Dense and Erect Panicle 1 This encodes an atypical plant G protein γ subunit with a cysteine-rich region at its C-terminus. In natural variations, deletion of the C-terminus of the dep1-1 protein enhances apical meristem activity, resulting in increased branch number and grain number per spike. In NIL- dep1-1 middle OsCKX2 ( Cytokinin oxidase / dehydrogenase 2 The expression level of ) was significantly downregulated, suggesting that it may be due to enhanced cytokinin function, thereby promoting spikelet branching. dep1-1 While increasing the number of grains per panicle, it reduces the sensitivity of rice growth to nitrogen fertilizer, enhances nitrogen assimilation capacity, and suppresses the nitrogen response of grain number. Even under low-nitrogen conditions, it still produces a relatively high number of grains per panicle, achieving high yield with low nitrogen. Therefore, elucidating the functional mechanism of the synergistic regulation of rice grain number by dep1-1 and nitrogen fertilizer will contribute to breeding improvements that increase grain number under low-nitrogen conditions, potentially improving both crop yield and nitrogen fertilizer use efficiency simultaneously. Summary of the Invention

[0005] The purpose of this invention is to provide the application of rice OsTMK1 protein or its truncated form and encoding gene in regulating the number of grains per panicle and nitrogen fertilizer use efficiency in plants.

[0006] To achieve the objectives of this invention, in a first aspect, any of the following applications of rice OsTMK1 protein or its truncated form, encoding gene, and related biological materials are made: 1) Used to regulate the number of grains per spike in plants; 2) Used to regulate the nitrogen fertilizer utilization efficiency of plants.

[0007] OsTMK1 The (Transmembrane Kinase 1) gene is identified as LOC_Os03g50810 in the Rice Gene Database (https: / / rice.uga.edu / index.shtml).

[0008] The OsTMK1 protein is: A. A protein consisting of the amino acid sequence shown in SEQ ID NO:1; or B. Proteins derived from A with one or more amino acids substituted, deleted, or added to the sequence shown in SEQ ID NO:1 and having equivalent functions.

[0009] The truncated form of the OsTMK1 protein is: a) A protein consisting of amino acid from amino acid position 526 to the C-terminal amino acid of the sequence shown in SEQ ID NO:1; or b. Proteins derived from a with one or more amino acids substituted, deleted, or added to the sequence shown in a, and having the same function.

[0010] Furthermore, the aforementioned regulation is positive regulation.

[0011] In this invention, the plant is a monocotyledonous plant or a dicotyledonous plant, preferably a grass, and more preferably rice (such as japonica rice).

[0012] Furthermore, the biological materials include, but are not limited to, recombinant DNA, expression cassettes, transposons, plasmid vectors, viral vectors, engineered bacteria, or transgenic cell lines.

[0013] Secondly, the present invention provides a method for increasing the number of grains per panicle while reducing the plant's sensitivity to nitrogen fertilizer, the method comprising: feeding rice... OsTMK1 Genes or their variants are introduced into plants via plasmids or integrated into plant chromosomes through genetic engineering.

[0014] Thirdly, the present invention provides a method for increasing the number of grains per panicle in rice under low nitrogen conditions, the method comprising: enhancing the number of grains per panicle in rice. OsTMK1 Expression of genes or their variants.

[0015] Furthermore, the enhancement can be selected from the following 1) to 6), or an optional combination thereof: 1) By importing a plasmid containing the gene or a variant thereof; 2) By increasing the copy number of the aforementioned gene or its variants on the rice chromosome; 3) By altering the promoter sequence of the aforementioned gene or its variant on the rice chromosome; 4) By operatively linking a strong promoter to the gene or a variant thereof; 5) By introducing enhancers; 6) Enhancement is achieved by using genes or alleles that encode the corresponding enzymes or proteins with high activity.

[0016] Expression vectors carrying the target gene can be introduced into plant cells using conventional biotechnological methods such as Ti plasmids, plant virus vectors, direct DNA transformation, microinjection, and electroporation (Weissbach, 1998, Method for Plant Molecular Biology VIII, Academy Press, New York, pp. 411-463; Geiserson and Corey, 1998, Plant Molecular Biology, 2). nd Edition).

[0017] Fourthly, the present invention provides the application of transgenic plants obtained according to the method in plant breeding.

[0018] Furthermore, breeding methods include, but are not limited to, transgenic, hybridization, backcrossing, self-pollination, or asexual reproduction.

[0019] The preferred methods for transgenic technology include: Ti plasmid, plant virus vector, direct DNA transformation, microinjection, gene gun, electroporation, or Agrobacterium-mediated transformation.

[0020] By employing the above technical solution, the present invention has at least the following advantages and beneficial effects: This invention is the first discovery of rice OsTMK1 Genes are related to the number of grains per ear and nitrogen fertilizer use efficiency in plants; enhancing these genes in plants can help. OsTMK1 The expression of this substance can significantly increase the number of grains per spike and nitrogen fertilizer use efficiency in plants. This invention provides... OsTMK1 Genes and their applications can be used to improve plant germplasm resources and breed new rice varieties with high yield and high nitrogen fertilizer utilization efficiency, which has important application value. Attached Figure Description

[0021] Figure 1 The phylogenetic tree and spatiotemporal expression pattern of OsTMK1 provided in Embodiment 1 of the present invention. (A) Phylogenetic tree analysis of TMK in Arabidopsis thaliana and rice. (B) Spatiotemporal expression pattern analysis of OsTMK1 and OsTKL1, with OsActin1 as the internal reference, and the values ​​are mean ± standard error (n = 3).

[0022] Figure 2 Phenotypic analysis of the OsTMK1 and OsTKL1 gene knockout lines under the WYJ7-DEP1 background provided in Example 1 of this invention, namely ostmk1, ostkl1 single mutant and ostmk1 ostkl1 double mutant; where (A) ear type, scale bar is 5cm; (B) number of primary branches; (C) number of secondary branches; (D) number of grains per ear. (BD) The values ​​in the figure are mean ± standard error (n = 8), different letters indicate significant differences (P < 0.05), and the significance analysis is performed using Duncan's test.

[0023] Figure 3 Phenotypic analysis of the transgenic lines overexpressing the OsTMK1 gene provided in Example 2 of this invention; where (A) ear type, scale bar is 5 cm; (B) number of primary branches; (C) number of secondary branches; (D) number of grains per ear. (BD) The values ​​in the figure are mean ± standard error (n = 8), different letters indicate significant differences (P<0.05), and the significance analysis was performed using Duncan's test.

[0024] Figure 4 Phenotypic analysis of the transgenic lines overexpressing the OsTKL1 gene provided in Example 2 of this invention; where (A) ear type, scale bar is 5 cm; (B) number of primary branches; (C) number of secondary branches; (D) number of grains per ear. (BD) The values ​​in the figure are mean ± standard error (n = 8), different letters indicate significant differences (P<0.05), and the significance analysis was performed using Duncan's test.

[0025] Figure 5 Phenotypic analysis of the OsTMK1C overexpressing transgenic line under the WYJ7-dep1-1 ostmk1 ostkl1 double mutant background provided in Example 3 of this invention; where (A) ear type, scale bar is 5cm; (B) number of grains per ear. The values ​​in (B) are mean ± standard error (n = 6), different letters indicate significant differences (P<0.05), and the significance analysis was performed using Duncan's test.

[0026] Figure 6This invention provides Example 4 of the study on the response of the number of grains per ear of the ostmk1 ostkl1 mutant to nitrogen under different nitrogen fertilizer conditions (low nitrogen 60 kg / ha and high nitrogen 210 kg / ha) in the WYJ7-dep1-1 background. (A) Ear type, scale bar is 5 cm. (B) Number of primary branches; (C) Number of secondary branches; (D) Number of grains per ear; (E) Nitrogen-mediated growth response (NGR) of grains per ear. (BE) The values ​​in the figures are mean ± standard error (n = 8), different letters indicate significant differences (P < 0.05), and the significance analysis used the Duncan test.

[0027] Figure 7 For the transgenic lines of WYJ7-DEP1 and overexpressing OsTMK1 and OsTMK1C provided in Example 5 of this invention, under different nitrogen fertilizer conditions (low nitrogen 60 kg / ha and high nitrogen 210 kg / ha), the nitrogen response ability of the number of grains per ear was statistically analyzed; where (A) ear type, scale bar is 5 cm; (B) number of primary branches; (C) number of secondary branches; (D) number of grains per ear; (E) nitrogen response ability of the number of grains per ear (NGR). (BE) The values ​​in the figure are mean ± standard error (n=8), different letters indicate significant differences (P<0.05), and the significance analysis is performed by Duncan's test. Detailed Implementation

[0028] This invention provides a gene that regulates the number of grains per panicle and nitrogen fertilizer use efficiency in rice, and its application.

[0029] In the early stages, the laboratory used radiation mutagenesis and genetic screening to obtain the mutant sod41, which exhibits a significant reduction in the number of grains per ear under the WYJ7 background carrying the dep1-1 allele. suppressor of dep1-1 grain numbers 41 The inventors obtained the target gene OsTKL2 (Transmembrane Kinase Like 2) for regulating grain number per panicle through map-based cloning. Phylogenetic analysis revealed two homologous genes for OsTKL2 in rice: OsTMK1 and OsTKL1. Genetic analysis demonstrated that both OsTMK1 and OsTKL1 genes regulate grain number per panicle in rice, and overexpression of the OsTMK1 gene and its intracellular region (OsTMK1C) increases grain number per panicle. Furthermore, the inventors demonstrated that overexpression of the OsTMK1 gene in rice increases grain number per panicle under low-nitrogen conditions. This research on the OsTMK1 gene will provide a theoretical basis for molecular design breeding that increases grain number per panicle and promotes nitrogen efficiency in crops.

[0030] In a first aspect, the present invention provides the application of OsTMK1 protein, or its encoding gene, or biological materials containing its encoding gene in regulating the number of grains per spike and nitrogen fertilizer use efficiency in plants.

[0031] The present invention further provides the application of OsTMK1 protein, or its encoding gene, or biological material containing its encoding gene in the cultivation of plants with high ear grain number and high nitrogen fertilizer utilization efficiency.

[0032] The present invention further provides the application of OsTMK1 protein, or its encoding gene, or biological materials containing its encoding gene in the improvement of plant germplasm resources.

[0033] Furthermore, the OsTMK1 protein comprises any one of the following amino acid sequences: (1) The amino acid sequence as shown in SEQ ID NO:1; (2) An amino acid sequence of a protein with the same function obtained by replacing, inserting or deleting one or more amino acids of the amino acid sequence shown in SEQ ID NO:1.

[0034] Furthermore, the gene encoding the OsTMK1 protein includes any one of the following nucleotide sequences: (1) The nucleotide sequence shown in SEQ ID NO:2; (2) A nucleotide sequence that encodes a protein with the same function, obtained by substituting, deleting or inserting one or more nucleotides into the nucleotide sequence shown in SEQ ID NO:2; (3) A nucleotide sequence that can hybridize with a nucleotide sequence such as SEQ ID NO:2 under strict conditions.

[0035] Furthermore, the plant is a monocotyledonous or dicotyledonous plant, preferably rice.

[0036] Furthermore, the biological material is an expression cassette, a vector, or a transgenic cell.

[0037] The transgenic cells described in this invention can be animal cells, plant cells, or microbial cells, preferably plant cells.

[0038] The transgenic cells described in this invention do not include transgenic cells that have the ability to develop independently into a complete individual, that is, they do not include plant varieties and animal varieties.

[0039] In a second aspect, the present invention provides a method for cultivating transgenic plants, comprising: Regulate the expression level of OsTMK1 protein in the plant; The amino acid sequence includes any one of the following: (1) The amino acid sequence as shown in SEQ ID NO:1; (2) An amino acid sequence of a protein with the same function obtained by replacing, inserting or deleting one or more amino acids of the amino acid sequence shown in SEQ ID NO:1.

[0040] Furthermore, the expression level of OsTMK1 protein in the plant can be regulated using any of the following methods: Genetically modified organisms (GMOs), hybridization, backcrossing, self-pollination, or asexual reproduction.

[0041] The preferred methods for transgenic technology include: Ti plasmid, plant virus vector, direct DNA transformation, microinjection, gene gun, electroporation, or Agrobacterium-mediated transformation.

[0042] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0043] Example 1: The function of OsTMKs in regulating the number of grains per ear is redundant. The inventors constructed a phylogenetic tree of the TMK protein family in rice and Arabidopsis thaliana using MEGA, and found that in addition to OsTKL2, rice also has two other TMK-type homologous proteins, OsTMK1 (LOC_Os03g50810) and OsTKL1 (LOC_Os04g58910), with amino acid sequences shown in SEQ ID NO:1 and SEQ ID NO:4. RNA was extracted from roots, seedling leaves, stem nodes, leaf sheaths, flag leaves, young panicles (0.2 cm), young panicles (3-6 cm), and young panicles (6-9 cm) of wild-type rice. The RNA was then converted to cDNA, and the expression levels of OsTMK1 and OsTKL1 genes in rice were detected using quantitative real-time PCR. Primers for quantitative detection were designed using the gene sequences (SEQ ID NO:2, SEQ ID NO:5) (Table 1).

[0044] Table 1 Primer sequences used for OsTMK1 and OsTKL1 gene expression analysis.

[0045] The results showed that the expression level of OsTMK1 was relatively high in various tissues or parts. This suggests that the OsTMK1 gene may be involved in regulating the development of young spikelets. Figure 1 , AB).

[0046] The inventors designed knockout vector primers using the OsTMK1 and OsTKL1 gene sequences (Table 2), ligated the target sequences into the sgRNA expression cassette (sequences SEQ ID NO:6, SEQ ID NO:7), and inserted pYLCRISPR / Cas9Pubi-MH (this vector was kindly provided by the laboratory of Liu Yaoguang, South China Agricultural University; see A Robust CRISPR / Cas9 System for Convenient, High-Efficiency Multiplex Genome Editing in Monocot and DicotPlants, Ma Xingliang et al., DOI:10.1016 / j.molp.2015.04.007) using Agrobacterium-mediated transformation. This yielded single knockout lines of ostmk1 and ostkl1, and double knockout lines of ostmk1 and ostkl1 in the WYJ7-DEP1 background. The results showed that, compared with the control WYJ7-DEP1, the spike type and number of grains per spike of the two single mutants, ostmk1 and ostkl1, were not significantly changed. However, the ostmk1-ostkl1 double mutant exhibited a significant phenotype with reduced primary and secondary branches, ultimately leading to a 24% reduction in the number of grains per spike. Figure 2 (AD).

[0047] The above results indicate that the OsTMK1 and OsTKL1 genes positively regulate the number of grains per panicle in rice and exhibit functional redundancy.

[0048] The inventors planted the rice in Hefei City, Anhui Province, and Lingshui Li Autonomous County, Hainan Province. After the plants matured, they counted traits such as the number of primary branches, the number of secondary branches, and the number of grains per panicle. The specific statistical method was as follows: after the rice matured in the field, the tallest single effective panicle was selected, and the number of primary branches, secondary branches, and grains per panicle on the main tiller were counted.

[0049] Table 2 Primer pairs used for constructing OsTMK1 and OsTKL1 gene knockout vectors

[0050] Example 2: Overexpression of OsTMK1 increases the number of grains per panicle in rice. The inventors extracted total RNA from the leaves of the WYJ7-DEP1 rice material, reverse transcribed it to synthesize cDNA, and used the cDNA as a template to perform PCR amplification of OsTMK1 using Do-OsTMK1-F and Do-OsTMK1-R primers (Table 3). The pActin::OsTMK1 vector was constructed by ligating the pCAMBIA2300 vector (purchased from Abogen (Shanghai) Trading Co., Ltd., catalog number ab275758) backbone using the Gateway BPClonase II Enzyme Mix and Gateway LR Clonase II Enzyme Mix kits (purchased from Invitrogen). The overexpression vector WYJ7-dep1-1 was transformed using Agrobacterium-mediated transformation to obtain positive transgenic plants. A line with an appropriate OsTMK1 overexpression fold (pActin::OsTMK1 3#) was selected and crossed with WYJ7-DEP1 to obtain homozygous OsTMK1 overexpression lines under the WYJ7-DEP1 background. Results showed that OsTMK1 overexpression under the WYJ7-DEP1 background significantly altered the panicle phenotype of rice. The OsTMK1 overexpression lines showed increased secondary branches and a 26% increase in grain number per panicle. Figure 3 (AD). The inventors designed Do-OsTKL1-F and Do-OsTKL1-R primers (Table 3) for PCR amplification of OsTKL1. Using the same method, they constructed the OsTKL1 overexpression vector pActin::OsTKL1-flag. Through genetic transformation and hybridization, homozygous OsTKL1 overexpression lines were obtained under the WYJ7-DEP1 background. The results showed that the number of grains per ear overexpressing OsTKL1 under the WYJ7-DEP1 background was not significantly different from the control. Figure 4 (AD).

[0051] This experiment shows that OsTMK1 is a positive regulator of grain number in rice panicles, and overexpression of this gene can increase the number of grains per panicle.

[0052] Table 3 Primer pairs used for constructing the OsTMK1 gene overexpression vector.

[0053] Example 3: Overexpression of the OsTMK1C gene increases the number of grains per ear. Studies in Arabidopsis thaliana have reported that AtTMK1 has the function of cleaving its intracellular region to regulate downstream factors. To investigate whether the intracellular region of rice OsTMK1 has biological functions and its effect on regulating panicle grain number, different OsTMK1 overexpression lines were constructed. The inventors constructed an OsTMK1 overexpression line in the WYJ7-dep1-1 ostmk1 ostkl1 double mutant background. mutTransgenic material of OsTMK1C. OsTMK1 mut This refers to the deletion of 19 amino acids (524 to 526 and 541 to 556) of the mutant OsTMK1 protein, as reported in the reference research, to simulate the inability to cleave the C-terminus. The intracellular region of OsTMK1 (OsTMK1C) is the C-terminal fragment of the OsTMK1 protein from 526 to 962 (SEQ ID NO:1).

[0054] The inventors designed OsTMK1 based on the OsTMK1 gene cDNA sequence (SEQ ID NO: 2). mut The OsTMK1C overexpression vector primers (Table 4) were used for PCR amplification to obtain OsTMK1. mut And OsTMK1C. Using Gateway BP Clonase II Enzyme Mix and Gateway LR Clonase II Enzyme Mix kits, the gene was ligated into the pCAMBIA2300 vector backbone. The 3007 bp OsTMK1 gene promoter sequence (SEQ ID NO:3) was selected to construct pOsTMK1::OsTMK1. mut The pOsTMK1::OsTMK1C (single intracellular region) vector, pActin::OsTMK1C-flag vector, and Agrobacterium-mediated transformation were used to transform the WYJ7-dep1-1 ostmk1 ostkl1 double mutant, respectively, to obtain positive transgenic plants. Statistical analysis revealed that the simulated non-cleavable form of OsTMK1... mut Transgenic materials overexpressing OsTMK1C could not revert to the grain number phenotype, while transgenic materials overexpressing OsTMK1C driven by their own promoter and Actin promoter could increase secondary branches, thus partially reverting to the reduced grain number phenotype of the WYJ7-dep1-1 ostmk1 ostkl1 double mutant. Figure 5 , AB).

[0055] This experiment demonstrates that OsTMK1C has a biological function of promoting the number of grains per ear.

[0056] Table 4 OsTMK1 mut Primer pairs used for constructing the OsTMK1C overexpression vector

[0057] Example 4: The number of grains per ear in the ostmk1 and ostkl1 mutants is relatively insensitive to nitrogen fertilizer. The above examples demonstrate that the rice ostmk1 ostkl1 double mutant is a dep1-1 repressor with a significant reduction in grain number per panicle. To study the nitrogen response of grain number per panicle after the OsTMK1 OsTKL1 mutation, the inventors planted WYJ7-DEP1, WYJ7-dep1-1, and WYJ7-dep1-1 ostmk1 ostkl1 in the field with different nitrogen application rates (60 kg / ha for low nitrogen and 210 kg / ha for high nitrogen). Statistical analysis revealed that the panicle phenotype of WYJ7-DEP1 showed a significant nitrogen response, with a 28% reduction in grain number per panicle under low nitrogen fertilizer conditions; WYJ7-dep1-1 only reduced the grain number per panicle by 15% under low nitrogen conditions, and was still higher than the grain number per panicle of WYJ7-DEP1 under high nitrogen conditions, indicating that dep1-1 reduces the sensitivity of grain number per panicle to nitrogen fertilizer and still has a high grain number per panicle under low nitrogen conditions. Regardless of whether the growing conditions were high or low nitrogen, the number of branches and grains per spike of WYJ7-dep1-1 ostmk1 ostkl1 showed little change, with the number of grains per spike decreasing by 11% under low nitrogen conditions. Figure 6 (AD).

[0058] To describe the ability of rice grain number to respond to nitrogen levels, the NGR (Nitrogen-mediated Growth Response) was introduced = (Number of grains per panicle under high nitrogen - Number of grains per panicle under low nitrogen) / Number of grains per panicle under low nitrogen. The NGR value can be used as a standard to measure the nitrogen sensitivity of rice grain number. Compared with WYJ7-DEP1, the NGR values ​​of grain number per panicle in WYJ7-dep1-1 and WYJ7-dep1-1ostmk1ostkl1 were significantly reduced. Although the number of grains per panicle was significantly reduced under both high and low nitrogen conditions, the mutants exhibited a relatively insensitive response to nitrogen fertilizer level changes, more similar to dep1-1. Figure 6 E).

[0059] This experiment shows that OsTMK1 is involved in regulating the rice panicle grain number response to nitrogen fertilizer.

[0060] Example 5: Overexpression of OsTMK1 and OsTKM1C increases grain number per ear under low nitrogen conditions. To further analyze the effects of OsTMK1 and OsTMK1C genes on rice panicle grain number and nitrogen fertilizer use efficiency, the inventors planted WYJ7-DEP1, WYJ7-DEP1 pActin::OsTMK1, and WYJ7-DEP1 pActin::OsTMK1C-flag in fields with different nitrogen application rates (60 kg / ha for low nitrogen and 210 kg / ha for high nitrogen). Statistical analysis of panicle phenotypes revealed that transgenic lines overexpressing OsTMK1 and OsTMK1C showed significantly increased primary and secondary branches and panicle grain number. The panicle grain number under low nitrogen conditions was close to that of the wild type under high nitrogen conditions. Figure 7 Compared with WYJ7-DEP1, the NGR values ​​of grain number per ear in OsTMK1 and OsTMK1C overexpression lines were significantly reduced (AD). Figure 7 The results indicate that OsTMK1 is an important new gene regulating the nitrogen response of grain number in rice panicles, and that overexpression of OsTMK1 and OsTMK1C has a more significant effect on increasing grain number under low nitrogen conditions.

[0061] In summary, OsTMK1 is conserved in its functions of regulating grain number per panicle and nitrogen fertilizer use efficiency in rice, exhibiting functional redundancy. Overexpression of both OsTMK1 and OsTMK1C genes can increase grain number per panicle and nitrogen fertilizer use efficiency in rice. These experimental results demonstrate that the OsTMK1 gene has the function of increasing grain number per panicle and improving nitrogen fertilizer use efficiency in rice, and has broad application prospects in high-yield and nitrogen-efficient rice breeding.

[0062] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. Any of the following applications of rice OsTMK1 protein or its truncated form, encoding gene, and related biological materials: 1) Used to regulate the number of grains per spike in plants; 2) Used to regulate the nitrogen fertilizer use efficiency of plants; The OsTMK1 protein is: A. A protein consisting of the amino acid sequence shown in SEQ ID NO:1; or B. Proteins derived from A with one or more amino acids substituted, deleted, or added to the sequence shown in SEQ ID NO:1 and having the same function; The truncated form of the OsTMK1 protein is: a) A protein consisting of amino acid from amino acid position 526 to the C-terminal amino acid of the sequence shown in SEQ ID NO:1; or b. Proteins derived from a with one or more amino acids substituted, deleted, or added to the sequence shown in a, and having the same function.

2. The application according to claim 1, characterized in that, The regulation mentioned is a positive regulation.

3. The application according to claim 1 or 2, characterized in that, The plant is a monocotyledonous or dicotyledonous plant, preferably a grass, and more preferably rice.

4. The application according to any one of claims 1-3, characterized in that, The biological material is recombinant DNA, expression cassette, transposon, plasmid vector, viral vector, or engineered bacteria.

5. A method for increasing the number of grains per spike while reducing the plant's sensitivity to nitrogen fertilizer, characterized in that, The method includes: [the process involves] rice... OsTMK1 Genes or their variants are introduced into plants via plasmids or integrated into plant chromosomes through genetic engineering. The OsTMK1 The gene encodes the rice OsTMK1 protein as described in claim 1; OsTMK1 The variant of the gene encodes a truncated form of the rice OsTMK1 protein as described in claim 1.

6. The method according to claim 5, characterized in that, The plant is a monocotyledonous or dicotyledonous plant, preferably a grass, and more preferably rice.

7. A method for increasing the number of grains per panicle in rice under low nitrogen conditions, characterized in that, The method includes: enhancing the rice OsTMK1 Expression of genes or their variants; The OsTMK1 Gene, OsTMK1 The variant of the gene is as described in claim 5.

8. The method according to claim 7, characterized in that, The enhancement method is selected from the following 1) to 6), or any combination thereof: 1) By importing a plasmid containing the gene or a variant thereof; 2) By increasing the copy number of the aforementioned gene or its variants on the rice chromosome; 3) By altering the promoter sequence of the aforementioned gene or its variant on the rice chromosome; 4) By operatively linking a strong promoter to the gene or a variant thereof; 5) By introducing enhancers; 6) Enhancement is achieved by using genes or alleles that encode the corresponding enzymes or proteins with high activity.

9. The use of transgenic plants obtained by the method according to any one of claims 5-8 in plant breeding.

10. The application according to claim 9, characterized in that, Breeding methods include transgenic, hybridization, backcrossing, self-pollination, or asexual reproduction.

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