Application of a protein for regulating nitrogen utilization rate of rice and method for regulating nitrogen utilization rate of rice

Abstract

The present invention provides an application of a protein for regulating nitrogen utilization rate of rice and a method for regulating nitrogen utilization rate of rice, belonging to the field of biotechnology, and specifically discloses an application of rice OsUBP15 protein in positively regulating nitrogen utilization rate in response to nitrogen, and a method for regulating nitrogen utilization rate of rice. OsUBP15 After the gene is overexpressed, rice will become insensitive to nitrogen and reduce the nitrogen absorption efficiency of rice. OsUBP15 Genetically improving the activity of OsUBP15 protein can significantly increase the nitrogen absorption efficiency of rice, which helps to understand the molecular mechanism of plant response to nitrogen to regulate nitrogen utilization, and provides an effective target for improving nitrogen utilization efficiency of crops such as rice.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, relates to plant transgenic biotechnology breeding, and particularly relates to the application of a protein for regulating rice nitrogen utilization rate and a method for regulating rice nitrogen utilization rate. Background Art

[0002] Rice ( Oryza sativa L. ) is an important food crop widely planted in the world, and more than half of the world's population relies on rice as their staple food. As the world's population increases year by year, the rigid demand for food continues to increase, which puts forward severe requirements for increasing crop yields. The application of chemical fertilizers is an important driving force for increasing the yield of food crops such as rice. However, the large-scale application of chemical fertilizers has caused a series of environmental and ecological problems, which has seriously restricted the sustainable development of agriculture. Therefore, how to improve the utilization efficiency of crop fertilizers and not reduce the yield under the premise of reducing the application of chemical fertilizers is a huge challenge currently facing crop genetic breeding.

[0003] In agricultural production, nitrogen fertilizer is the most important fertilizer component to promote the increase of rice and other crops. Nitrogen metabolism, as one of the basic metabolic activities of plants, is related to the yield and quality traits of crops such as plant biomass, photosynthetic efficiency, tillering, seed size, etc. The nitrogen utilization rate of crops is regulated by an extremely conservative self-signaling network. In recent years, many conservative key genes and signals regulating the nitrogen utilization rate of plants have been discovered, which has attracted widespread attention. The conserved nitrate (NO 3- ) transporter NPF / NRT and ammonium (NH 4+ ) The transporter AMT is essential for nitrogen absorption and transport, and promotes nitrogen utilization efficiency of crops; key nitrogen assimilation enzymes such as nitrate reductase NR, glutamine / glutamate synthetase (GS / GOAT) cycle-related enzymes, asparagine synthetase AS and amino acid transporter AAP1 are also key signals for regulating nitrogen utilization efficiency; the 48bp base lost in the 10th intron of asparagine synthetase THP9 / ASN4 in wild maize ( THP9-T ) can significantly improve the nitrogen utilization rate of cultivated varieties; gibberellin repressor DELLA protein (such as green revolution gene SLR1 and Rht-1 ) interferes with the GRF4-GIF1 complex and inhibits nitrogen utilization; miR396 Target mutation ( GRF4 ngr2 ) increases its abundance and thus promotes nitrogen utilization; gibberellins also mediate nitrogen-induced increase in tiller number through degradation of NGR5 by its receptor GID1; Rht-B1b , EamA-B , ZnF-B Deletion of three linked genes ( r - e - z) inhibits brassinolide response and significantly improves nitrogen utilization efficiency; dominant mutation in G protein γ subunit DEP1 dep1-1 Significantly improve nitrogen utilization efficiency and yield under low nitrogen conditions; DEP1 interacts with the transcription factor MADS1 to promote nitrogen utilization, and another γ subunit GS3 that antagonizes DEP1 negatively regulates nitrogen utilization; in addition, a series of transcription factors such as OsTCP19 , NLP , NAC , Dof , TabZIP60 , TaNFYA-B1 It can also regulate nitrogen utilization, including OsTCP19 29 bp base insertion in the promoter region ( OsTCP19 H ) can significantly improve nitrogen utilization efficiency and is a key gene for adaptability of different rice varieties to regional soil nitrogen content. Although the discovery of these genes has made substantial contributions to the understanding of the regulatory mechanism of crop nitrogen utilization efficiency and the improvement of crop yields, the understanding of the genetic regulatory network of plant nitrogen fertilizer utilization efficiency is still very limited, and the discovery of new nitrogen utilization regulatory genes is still of great significance. Summary of the invention

[0004] In response to the above problems, the present invention provides an application of a protein for regulating the nitrogen utilization rate of rice and a method for regulating the nitrogen utilization rate of rice, which provides new insights into the molecular mechanism by which rice and other plants respond to external nitrogen levels to promote nitrogen absorption and utilization, and provides new genetic resources for improving rice with efficient nitrogen utilization.

[0005] To achieve the above purpose, the technical solution adopted by the present invention is:

[0006] An application of the above protein for regulating nitrogen utilization rate of rice, wherein the application is that the OsUBP15 protein synchronizes the external nitrogen level and the nitrogen absorption rate.

[0007] Further, the protein is a deubiquitinating enzyme OsUBP15 protein that responds to nitrogen and positively regulates nitrogen utilization throughout the growth cycle;

[0008] The Uniprots accession number of OsUBP15 protein is Q0E2F9, and the RefSeq_Locus accession number is XP_015627057;

[0009] OsUBP15 protein is a protein that synchronizes the external nitrogen level and nitrogen absorption rate in rice. The molecular mechanism of rice regulating nitrogen absorption efficiency in response to external nitrogen levels has been preliminarily analyzed: after sensing the external nitrogen level, rice adjusts the OsUBP15 protein level to determine the nitrogen absorption rate, thereby ensuring the growth of plants under different nitrogen levels. This is a new molecular mechanism for plants to respond to external nitrogen levels and coordinate nitrogen absorption and utilization efficiency.

[0010] Furthermore, the nitrogen utilization rate is positively regulated in response to nitrogen throughout the growth cycle, including at least one of the following situations:

[0011] At the seedling stage, nitrogen promotes the increase of aboveground dry weight;

[0012] During the vegetative growth period, nitrogen promotes plant growth and increases photosynthetic rate;

[0013] During the reproductive growth period, nitrogen promotes seed enlargement;

[0014] The OsUBP15 protein positively regulates the nitrogen uptake rate;

[0015] Nitrogen promotes the accumulation of OsUBP15 protein.

[0016] The gene encoding the above OsUBP15 protein is OsUBP15 Gene;

[0017] OsUBP15 The gene MSU_Locus number is LOC_Os02g14730, the NCBI_Locus number is LOC4328857, and the RAP_Locus number is Os02g0244300;

[0018] The function of the gene encoding the above OsUBP15 protein is OsUBP15 The gene positively regulates the function of nitrogen utilization efficiency;

[0019] The functions include knocking out OsUBP15 Gene, which significantly reduces rice's sensitivity to nitrogen at different growth stages;

[0020] in, osubp15 The gene editing knockout homozygous mutant was insensitive to the increase in aboveground parts after nitrogen application at the seedling stage, insensitive to the increase in growth and photosynthetic rate promoted by nitrogen during the vegetative growth period, and insensitive to the increase in seed size promoted by nitrogen during the reproductive growth period. The specific manifestations are:

[0021] At the seedling stage, compared with the wild type, osubp15 The gene-edited knockout homozygous mutant showed a small increase in aboveground biomass after nitrogen application;

[0022] In the vegetative growth stage, compared with the wild type, osubp15 The gene-edited knockout homozygous mutant grew slowly after nitrogen application;

[0023] In the vegetative growth stage, compared with the wild type, osubp15 The photosynthetic rate of the homozygous mutant knocked out by gene editing increased slightly after nitrogen application;

[0024] In the reproductive growth stage, compared with the wild type, osubp15 The gene-edited knockout homozygous mutant had a small increase in seed size after nitrogen application;

[0025] Knockout OsUBP15 Gene can significantly reduce the nitrogen absorption efficiency of rice, osubp15 Gene editing knocks out homozygous mutant absorption 15 The rate of N-labeled NH4NO3 decreased significantly compared with the wild type;

[0026] Said OsUBP15 Gene knockout is performed using any of the following techniques:

[0027] Knockout using T-DNA insertion OsUBP15 Gene;

[0028] Knockout using gene editing OsUBP15 Gene;

[0029] Knockout by chemical mutagenesis OsUBP15 Gene;

[0030] Knockout by physical mutagenesis OsUBP15 Gene;

[0031] The function also includes overexpression OsUBP15 The gene may enhance the activity of OsUBP15 protein to improve the nitrogen utilization efficiency of rice;

[0032] Overexpression OsUBP15 Genes or methods for increasing the activity of OsUBP15 protein include any of the following:

[0033] Overexpression through a strong promoter OsUBP15 Gene;

[0034] Enhanced by mutations in promoter, UTR and other regions OsUBP15 Gene expression;

[0035] The activity of OsUBP15 protein is enhanced through gene mutation and other means.

[0036] Furthermore, the gene encoding the above OsUBP15 protein is OsUBP15 Gene;

[0037] Said OsUBP15 The gene positively regulates nitrogen utilization. Furthermore, the application is to improve the nitrogen utilization of rice by overexpressing the OsUBP15 gene or increasing the activity of the OsUBP15 protein;

[0038] That is, the OsUBP15 protein fused to green fluorescent protein (GFP) overexpressed by a strong promoter (OsUBP15-GFP) accumulated in low amounts under nitrogen deficiency conditions but accumulated with increasing nitrogen levels;

[0039] Furthermore, overexpression OsUBP15 Genes or methods for increasing the activity of OsUBP15 protein include any of the following:

[0040] Overexpression of the OsUBP15 gene through a strong promoter;

[0041] The expression of OsUBP15 gene was enhanced by mutations in promoter, UTR and other regions;

[0042] Enhance the activity of OsUBP15 protein by gene mutation or other methods;

[0043] The present invention adopts the overexpression strain OsUBP15OE and cultivates it under nitrogen levels of 0N, 1 / 8N, 1 / 4N, 1 / 2N and 1N for 3 days, and then observes the root UBP15-GFP protein level under a fluorescence microscope. The UBP15-GFP protein level gradually accumulates with the increase of nitrogen level.

[0044] At the same time, the application can also be achieved by knocking out / knockdown OsUBP15 genes to reduce nitrogen use efficiency;

[0045] That is, by knocking out OsUBP15 Gene, acquired osubp15 Gene editing is used to knock out homozygous mutants, thereby reducing nitrogen absorption efficiency.

[0046] A method for regulating nitrogen utilization rate of rice, the method comprising planting OsUBP15 Gene overexpression strains or strains with enhanced OsUBP15 protein activity can regulate rice growth by controlling the planting environment.

[0047] Further, obtain OsUBP15 The gene overexpression strain or the strain with enhanced OsUBP15 protein activity includes any of the following:

[0048] Overexpression through a strong promoter OsUBP15 Genes to obtain OsUBP15 Gene overexpression strains;

[0049] Enhanced by mutations in promoter and UTR regions OsUBP15 Gene expression to obtain OsUBP15 Gene overexpression strains;

[0050] The activity of OsUBP15 protein is enhanced by gene mutation to obtain a strain with enhanced OsUBP15 protein activity.

[0051] Furthermore, regulating the planting environment means regulating the nitrogen level in the planting environment.

[0052] Further, the method is to plant OsUBP15 In the process of gene overexpression strains or strains with enhanced OsUBP15 protein activity, the nitrogen utilization efficiency is improved by regulating the nitrogen level in the planting environment.

[0053] The beneficial effects of the application of a protein for regulating the nitrogen utilization rate of rice and the method for regulating the nitrogen utilization rate of rice of the present invention are:

[0054] The present invention found that the OsUBP15 protein and its encoding gene both respond to nitrogen and positively regulate nitrogen utilization in different growth stages of rice; OsUBP15 The gene-edited knockout mutants were insensitive to nitrogen responses, including nitrogen-promoted increases in aboveground dry weight during the seedling stage, nitrogen-promoted growth and photosynthetic rate during the vegetative growth period, and nitrogen-promoted seed enlargement during the reproductive period. Further research found OsUBP15 The nitrogen absorption efficiency of the gene editing knockout mutant was significantly reduced, while overexpression of the OsUBP15 gene was able to improve nitrogen absorption efficiency. The OsUBP15 protein responded to nitrogen and accumulated as nitrogen levels increased. These results proved that the OsUBP15 protein and its encoding gene responded to nitrogen throughout the rice growth cycle and positively regulated the nitrogen utilization rate of rice, providing new insights into the molecular mechanism of how rice and other plants respond to external nitrogen levels to promote nitrogen absorption and utilization, and providing new gene resources for the improvement of nitrogen-efficient rice.

[0055] The present invention finds that knockout / knockdown OsUBP15 After the gene is overexpressed, rice will become insensitive to nitrogen and reduce the nitrogen absorption efficiency of rice. OsUBP15 Genetically improving the activity of OsUBP15 protein can significantly increase the nitrogen absorption efficiency of rice, which helps to understand the molecular mechanism of plant response to nitrogen to regulate nitrogen utilization, and provides an effective target for improving nitrogen utilization efficiency of crops such as rice. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Picture 1 In Example 1 of the present invention osubp15 Gene editing knockout homozygous mutants and osgrf4 Base comparison diagram of gene editing knockout homozygous mutant and wild-type ZH11; Figure a is osubp15 Base comparison diagram of gene editing knockout homozygous mutant and wild type ZH11, osubp15The gene editing knockout homozygous mutant is a homozygous mutant with GACC deleted and ACCAA inserted; Figure b is osgrf4 Base comparison diagram of gene editing knockout homozygous mutant and wild type ZH11, osgrf4 The gene editing knockout homozygous mutant is a homozygous mutant with missing AG;

[0057] Picture 2 In Example 2 of the present invention osubp15 The results of gene editing knockout homozygous mutants are insensitive to nitrogen response at the seedling stage; osubp15 The gene editing knockout homozygous mutant seedlings were less sensitive to nitrogen-induced increase in aboveground dry weight than wild-type ZH11 seedlings, which was consistent with the low nitrogen utilization efficiency. osgrf4 Gene editing knockout homozygous mutants are equivalent; Picture 2 In the table, 0N, 1 / 8N, 1 / 4N, 1 / 2N and 1N represent Yoshida rice culture medium (pH = 5.7) containing 0 mM, 0.157 mM, 0.313 mM, 0.625 mM and 1.25 mM NH4NO3, respectively. About 24 seedlings of different genotypes were tested under different nitrogen concentrations. ZH11 represents the wild type ZH11, osubp15 represent osubp15 Gene editing knocks out homozygous mutants, osgrf4 represent osgrf4 Gene editing knocks out homozygous mutants;

[0058] Picture 3 In Example 3 of the present invention osubp15 The results of gene editing knockout homozygous mutants being insensitive to nitrogen during the vegetative growth period; Figure a is osubp15 Comparison of the growth of the gene-edited knockout homozygous mutant and the wild-type ZH11 under different nitrogen conditions. osubp15 Compared with the wild-type ZH11, the growth of the homozygous mutant knocked out by gene editing is not significantly promoted by nitrogen application. Its growth trend is similar under the conditions of nitrogen fertilizer application (i.e. HN) and no nitrogen fertilizer application (i.e. LN); Figure b is osubp15 Comparison of photosynthetic rates of gene-edited knockout homozygous mutant and wild-type ZH11 under different nitrogen conditions. osubp15 The photosynthetic rate of the gene-edited knockout homozygous mutant was reduced compared with the wild-type ZH11 in response to nitrogen application. Five strains were tested at each nitrogen level for different genotypes. Picture 3 In the table, ZH11 represents wild-type ZH11. osubp15 represent osubp15 Gene editing knockout homozygous mutant, LN represents the condition without nitrogen fertilizer application, and HN represents the condition with nitrogen fertilizer application;

[0059] Picture 4In Example 4 of the present invention osubp15 The results of gene editing knockout homozygous mutants being insensitive to nitrogen during the reproductive growth period; Figure a is osubp15 Comparison of rice harvested under different nitrogen conditions between the gene-edited knockout homozygous mutant and the wild-type ZH11. osubp15 The seed enlargement of the homozygous mutant knocked out by gene editing was not significantly promoted by nitrogen application compared with the wild-type ZH11; Figure b is osubp15 Statistical results of 100-grain weight of the gene-edited knockout homozygous mutant and wild-type ZH11 under different nitrogen conditions. Grains harvested from three separate strains were tested under different genotype backgrounds and different treatments. Picture 4 In the table, ZH11 represents wild-type ZH11. osubp15 represent osubp15 Gene editing knockout homozygous mutant, LN represents the condition without nitrogen fertilizer application, and HN represents the condition with nitrogen fertilizer application;

[0060] Picture 5 The rice in Example 5 of the present invention OsUBP15 Comparison of gene-enhanced nitrogen absorption; Picture 5 In the table, ZH11 represents wild-type ZH11. osubp15 Representing the background of ZH11 osubp15 Gene editing knocks out homozygous mutants, osgrf4 Representing the background of ZH11 osgrf4 Gene editing knockout homozygous mutant, Kitaake represents wild-type Kitaake, lg1-1 Representing Kitaake's background OsUBP15 Gene mutant lg1-1 , OsUBP15OE represents the overexpression line OsUBP15OE in Kitaake background (ProUbi::gOsUBP15-GFP); Picture 5 Figures a and c in the figure are the phenotypes of seedlings grown under 1N hydroponic solution for 10 days; Picture 5 Figures b and d in the figure are from the rice seedlings grown at 1N for 10 days, transferred to Yoshida rice culture medium with 1.25mM 15N-labeled NH4NO3 for 3h, and after CaSO4 washing, the seedlings were dried and ground, and the isotope mass spectrometer was used to detect the 15 Results at the N level, where each genotype was replicated three times;

[0061] Picture 61 is a result diagram of the accumulation of rice OsUBP15 protein with increasing nitrogen levels in Example 6 of the present invention; wherein, Figure a is a comparison result diagram of the root UBP15-GFP protein level of the overexpression strain OsUBP15OE under different nitrogen levels under a fluorescence microscope, the left side is the fluorescence accumulation in the root apical meristem, and the right side is the fluorescence accumulation in the root mature zone; Figure b is a comparison result diagram of the root apical UBP15-GFP protein level of the overexpression strain OsUBP15OE under different nitrogen levels; Picture 6 In the table, 0N, 1 / 8N, 1 / 4N, 1 / 2N and 1N represent Yoshida rice culture medium (pH=5.7) containing 0mM, 0.157mM, 0.313mM, 0.625mM and 1.25mM NH4NO3, respectively. Pro Ubi ::gOsUBP15-GFP The overexpression line OsUBP15OE represents the Kitaake background; Picture 6 In this study, the overexpression line OsUBP15OE rice was cultured under different nitrogen levels for 7 days, and the root UBP15-GFP protein level was observed under a fluorescence microscope. The UBP15-GFP protein level gradually accumulated with the increase of nitrogen level. DETAILED DESCRIPTION

[0062] The technical solutions in the embodiments of the present invention are described clearly and completely below. In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention can also be implemented in other ways different from those described herein, and those skilled in the art can make similar generalizations without violating the connotation of the present invention, so the present invention is not limited by the specific embodiments disclosed below. The present invention is further described in detail below in conjunction with specific embodiments so that those skilled in the art can understand.

[0063] In addition, the experimental methods in the specific examples disclosed below are all conventional methods unless otherwise specified, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0064] In the following examples, rice with the ZH11 background osubp15 Gene editing knockout homozygous mutant (Accession No. BG101910A02) and osgrf4 The gene editing knockout homozygous mutant (germ number: BG101948A06) was purchased from Biogle (http: / / biogle.cn / ), Kitaake background osubp15 Mutants lg1-1 (Right now osubp15Gene editing knockout mutant) and overexpression line OsUBP15OE (OsUBP15-GFP#41, Pro Ubi ::gOsUBP15-GFP ,Right now OsUBP15 The gene overexpression strains were donated by the team of Academician Wan Jianmin from the Chinese Academy of Agricultural Sciences (recorded in the literature Shi, C., Ren, Y., Liu, L., Wang, F., Zhang, H., Tian, ​​P., Pan, T., Wang, Y., Jing, R., Liu, T., et al. (2019). Ubiquitin specific protease 15 has an important role inregulating grain width and size in rice. Plant Physiol 180, 381-391).

[0065] The data in the following examples were analyzed using GraphPad Prism 8 software, using Dunnett's multiple comparison test in One-Way ANOVA for single-factor data and Tukey's multiple comparison test in Two-Way ANOVA for two-factor data.

[0066] Example 1 No T-DNA insertion osubp15 Obtaining homozygous mutants of gene editing knockout and homozygous mutants of osgrf4 gene editing knockout

[0067] 1) Order mutants

[0068] Order ZH11 rice background from Biogen OsUBP15 (LOC_Os02g14730) gene-edited mutants (accession numbers: BG101910A02) and OsGRF4(LOC_Os02g47280) gene-edited mutants (germplasm number: BG101948A06), which were obtained using the CRISPR-Cas9 system (the acquisition method is described in Lu, Y., Ye, X., Guo, R., Huang, J., Wang, W., Tang, J., Tan, L., Zhu, JK, Chu, C., and Qian, Y. (2017). Genome-wide Targeted Mutagenesis in Rice Using theCRISPR / Cas9 System. Mol Plant 10, 1242-1245.), among which osubp15 The editing target of the gene editing mutant is 5'-ATGGGTCTTAAAGAAGGTCCTGG-3', located OsUBP15 the first exon of the gene (LOC_Os02g14730); osgrf4 The editing target of the gene editing mutant is 5'-AAGAAGCTGGACCCAGAGCCAGG-3', located OsGRF4 (LOC_Os02g47280) the second exon of the gene.

[0069] 2) Obtaining gene editing knockout materials without T-DNA insertion

[0070] To obtain a T-DNA-free osubp15 Gene editing knockout homozygous mutants and osgrf4 For the knockout mutant seeds, we first designed and synthesized primer pairs for the hygromycin resistance gene in T-DNA (i.e., hygromycin gene primer pairs): 5'-GAAGTGCTTGACATTGGGGAGT-3' and 5'-AGATGTTGGCGACCTCG TATT-3', extracted the DNA polymerase chain reaction (PCR) templates of these knockout mutants, and used the hygromycin gene primer pairs for PCR amplification to select strains without hygromycin gene amplification bands. OsUBP15 The target-specific amplification primer pairs were 5'-AGCTTTGGCCTGAGAGATCG-3' and 5'-GGCAACTTGAGGAGTGGCAA-3' and OsGRF4 The target-specific amplification primer pairs 5'-CTCGTGCTCGCTTCACGATTAG-3' and 5'-GCCTGTTCCACCACCAACAG-3' were used to amplify the T-DNA-free osubp15 Target fragments and osgrf4The target fragment in the mutant was sent to Qingke Biotechnology Co., Ltd. for sequencing to obtain the gene editing knockout homozygous strain (i.e. osubp15 Gene editing knockout homozygous mutants and osgrf4 Gene editing knockout homozygous mutant). Among them, osubp15 The gene editing knockout homozygous mutant is a homozygous mutant with GACC deleted and ACCAA inserted, such as Picture 1 As shown in Figure a; osgrf4 The gene editing knockout homozygous mutant is a homozygous mutant lacking AG, such as Picture 1 As shown in Figure b.

[0071] Then, for T-DNA-free osubp15 Gene editing knockout homozygous mutants and osgrf4 Gene editing knocked out homozygous mutants for breeding and obtained osubp15 Gene editing knockout homozygous mutant seeds and osgrf4 Gene editing knocks out homozygous mutant seeds.

[0072] Example 2 osubp15 Gene editing knockout homozygous mutants and osgrf4 Analysis of nitrogen sensitivity of seedlings of homozygous knockout mutants using gene editing

[0073] Wild-type ZH11 seeds (positive control), osubp15 Gene editing knockout homozygous mutant seeds (experimental group) and osgrf4 The seeds of the gene-edited knockout homozygous mutant (negative control) were soaked in tap water and cultured for 2 days at 28°C, 12h light / 12h dark for germination. The germinated corresponding seedlings were then transplanted into hydroponic boxes containing hydroponic solutions with different nitrogen concentrations for hydroponic culture, and new corresponding hydroponic solutions were replaced every day. Among them, the nitrogen levels in the hydroponic solutions with different nitrogen concentrations were 0N, 1 / 8N, 1 / 4N, 1 / 2N and 1N, that is, Yoshida rice culture solution (pH=5.7) containing NH4NO3 with concentrations of 0mM, 0.157mM, 0.313mM, 0.625mM, and 1.25mM. There were 24 seedlings per genotype in each treatment. After 8 days of hydroponic culture, the aboveground parts of the seedlings were collected, dried in an oven at 65°C, and the dry weight of the aboveground parts of the plants was measured. The results are as follows Picture 2 As shown in the figure, the aboveground part of wild-type ZH11 seedlings increased linearly with increasing nitrogen levels, but osubp15 Gene editing knockout homozygous mutant seedlings and osgrf4 The nitrogen-induced increase in aboveground dry weight in the seedlings of the gene-edited knockout homozygous mutant was smaller than that in the wild-type ZH11 seedlings, and under different nitrogen levels osubp15The dry weight of the aboveground part of the gene-edited knockout homozygous mutant seedlings was lower than that of the wild-type ZH11 seedlings under the same conditions, especially under higher nitrogen conditions (1 / 4N, 1 / 2N and 1N), the difference between the two was more significant. osubp15 Gene editing knockout homozygous mutants are sensitive to nitrogen and insensitive to nitrogen osgrf4 Gene editing knockout homozygous mutants are almost identical, indicating OsUBP15 Loss of gene function can significantly reduce the nitrogen use efficiency of rice seedlings.

[0074] Example 3 osubp15 Analysis of nitrogen sensitivity during the vegetative growth period of homozygous gene editing knockout mutants

[0075] The wild-type ZH11 seedlings and osubp15 The homozygous mutant seedlings were planted in small pots, and then transplanted into large rectangular pots filled with sandy soil after the seedlings grew up. osubp15 Two plants of each gene editing knockout homozygous mutant were planted, with a total of 6 pots. Among them, 3 pots were fertilized with nitrogen fertilizer (marked as HN) once a week, and 1L of 1.25mM NH4NO3 aqueous solution was applied each time; the other 3 pots were not fertilized with nitrogen fertilizer and served as the control group (marked as LN). The rice in these pots was placed in Sanya during the southern breeding season (November to March of the following year) under open-air conditions (temperature 22~30℃, about 12h light / 12h dark) and cultured for 3 weeks. The results are as follows Picture 3 As shown. Picture 3 As can be seen from Figure a, the wild type ZH11 is sensitive to nitrogen. Under the condition of nitrogen fertilizer (i.e. HN), the growth of the wild type ZH11 is significantly more vigorous than that under the condition of no nitrogen fertilizer (i.e. LN); osubp15 The gene editing knockout homozygous mutant showed a significant decrease in sensitivity to nitrogen, and there was no significant difference in growth between the application of nitrogen fertilizer (i.e., HN) and the absence of nitrogen fertilizer (i.e., LN). Furthermore, the photosynthetic rate of the fully expanded rice flag leaf was measured using a portable photosynthetic meter (LCi-T). The results were as follows: Picture 3 As shown in Figure b, the photosynthetic rate of wild-type ZH11 increased by 275% under nitrogen fertilizer (i.e., HN) conditions compared to the condition without nitrogen fertilizer (i.e., LN). osubp15 The photosynthetic rate increase of the homozygous mutant knocked out by gene editing was only 114%, which was significantly lower than that of the wild-type ZH11, further proving that OsUBP15 Gene knockout will reduce the nitrogen utilization rate and sensitivity of rice, making it impossible for nitrogen to significantly promote nutritional growth and increase photosynthetic rate.

[0076] Example 4 osubp15 Analysis of nitrogen sensitivity during reproductive growth of homozygous knockout mutants by gene editing

[0077] OsUBP15 Genes are involved in the regulation of seed size, and nitrogen plays a key role in promoting seed enlargement. osubp15 Whether the effect of nitrogen on seed size is mediated by gene editing knockout homozygous mutants, wild-type ZH11 and osubp15 The gene-edited knockout homozygous mutants were transplanted into rectangular pots filled with sandy soil, with 2 plants of each genotype planted in each pot, for a total of 6 pots. Among them, 3 pots were applied with nitrogen fertilizer (marked as HN) once a week for a total of 5 times, and each time 1L of 1.25mM NH4NO3 aqueous solution was applied; the other 3 pots were not applied with nitrogen fertilizer, but only irrigated with water, as a control group (marked as LN). The rice in these pots was cultured under open-air conditions (temperature 22~30℃, about 12h light / 12h dark) in Sanya during the southern breeding season (November to March of the following year). The rice was harvested after it matured. After the harvested rice was dried in an oven at 37℃, the rice phenotype was photographed and the weight of 100 grains of rice was weighed. The results are as follows Picture 4 As shown. Picture 4 It can be seen that the application of nitrogen fertilizer can significantly promote the grain size (seed width) and 100-grain weight of wild-type ZH11 rice. Among them, the 100-grain weight increased by 46.2% under the condition of applying nitrogen fertilizer (i.e. HN) compared with the condition without nitrogen fertilizer (i.e. LN). osubp15 Gene editing knockout homozygous mutants, under the same nitrogen application conditions, did not significantly promote osubp15 The rice grains of the homozygous mutants were enlarged and increased significantly under nitrogen fertilizer (HN) conditions compared with those without nitrogen fertilizer (LN). osubp15 The 100-grain weight of the homozygous mutant rice with gene editing only increased by 14.6%, which was significantly lower than the increase in wild-type ZH11 rice. These results show that OsUBP15 The gene is a key gene in rice's response to nitrogen to promote rice grain enlargement, and it plays an important role in improving the nitrogen utilization efficiency of rice during the reproductive growth period.

[0078] In summary, from the seedling stage, vegetative growth stage to reproductive growth stage, OsUBP15 Genes play a key role in regulating nitrogen utilization. OsUBP15 The gene significantly reduced nitrogen use efficiency of rice throughout the growth cycle, suggesting that other forms of OsUBP15 Genetic modification resulting in loss of function can also reduce the nitrogen utilization efficiency of rice throughout its growth cycle, including T-DNA insertion, physical mutagenesis and chemical mutagenesis.

[0079] Example 5 OsUBP15 Study on the regulatory effect of genes on nitrogen absorption efficiency

[0080] Although OsUBP15The gene positively regulates nitrogen use efficiency, but whether it regulates nitrogen absorption efficiency remains unknown. osubp15 Gene editing knockout homozygous mutant, ZH11 background osgrf4 Gene editing knockout homozygous mutant, wild-type Kitaake, mutant in Kitaake background lg1-1 (Right now osubp15 Gene editing knockout mutant), overexpression strain OsUBP15OE in Kitaake background ( Pro Ubi ::gOsUBP15- GFP ) seedlings were cultured in Yoshida rice culture medium (pH=5.7) containing 1.25 mM NH4NO3 for 10 days. The growth results showed that osubp15 The seedling growth of the homozygous mutant knocked out by gene editing was significantly worse than that of the wild-type ZH11, and the plants were shorter, which was related to the weakened nitrogen absorption. osgrf4 Gene editing knockout homozygous mutants are similar (e.g. Picture 5 Similarly, another OsUBP15 Gene mutant lg1-1 The growth of the seedlings of the overexpressing strain OsUBP15OE was also weaker than that of the wild-type Kitaake, while the growth of the seedlings of the overexpressing strain OsUBP15OE was significantly better than that of the wild-type Kitaake ( Picture 5 As shown in Figure c), it implies OsUBP15 Genes can promote nitrogen absorption and utilization. Further, the seedlings were transferred to Yoshida rice culture medium (pH = 5.7) with a concentration of 1.25mM 15N-labeled NH4NO3 for 3 hours, and then the seedling roots were washed with a 0.1mM calcium sulfate aqueous solution (CaSO4). After the seedlings were placed in a 65°C oven for drying, they were fully ground with a mortar and passed through a 100-mesh sieve. The resulting tissue dry powder was sent to the Environmental Stable Isotope Laboratory of the Chinese Academy of Agricultural Sciences to test the 15N content in the tissue (isotope mass spectrometer Isoprime-100 detection). The results are as follows Picture 5 As shown in Figure b and Figure d. Picture 5 As can be seen from Figure b, osubp15 The 15N absorption rate of the homozygous mutant knocked out by gene editing was significantly lower than that of the wild-type ZH11, which was consistent with the decreased nitrogen absorption rate. osgrf4 The knockout results were consistent with homozygous mutants. lg1-1 (Right now osubp15 The nitrogen absorption rate of the gene-edited knockout mutant was also significantly reduced compared with the wild-type Kitaake, while the overexpression line OsUBP15OE ( Pro Ubi ::gOsUBP15-GFP) had a significantly higher nitrogen absorption rate than the wild-type Kitaake ( Picture 5 These results show that OsUBP15 The gene positively regulates the rate of nitrogen uptake.

[0081] Considering osubp15 The gene editing knockout homozygous mutant showed decreased nitrogen sensitivity and nitrogen absorption rate, while the overexpression strain OsUBP15OE enhanced the nitrogen absorption rate. These results indicate that overexpression of a strong promoter, gene editing OsUBP15 Gene promoter or UTR, mutation enhancement of OsUBP15 protein function can effectively improve nitrogen absorption rate and nitrogen use efficiency, and provide a reference for rice and other crops containing nitrogen. UBP15 Gene homologs provide target genes for improving nitrogen utilization efficiency in plants.

[0082] Example 6 Detection of OsUBP15 protein response to nitrogen changes

[0083] Furthermore, the overexpression line OsUBP15OE in Kitaake background was further investigated. Pro Ubi ::gOsUBP15-GFP ) was studied in response to nitrogen in the protein level of OsUBP15-GFP in the overexpression line. After germination, the seeds of the overexpression line OsUBP15OE were transferred to Yoshida rice culture medium containing different nitrogen concentrations, and fresh culture medium was replaced every day. Among them, the nitrogen levels in the Yoshida rice culture medium containing different nitrogen concentrations were 0N, 1 / 8N, 1 / 4N, 1 / 2N and 1N, that is, Yoshida rice culture medium (pH=5.7) containing NH4NO3 concentrations of 0mM, 0.157mM, 0.313mM, 0.625mM, and 1.25mM. After 7 days of cultivation, the roots were taken and the GFP protein levels in the root tip and mature zone were observed under a fluorescence microscope (Olympus BX53). The results are shown in Picture 6 As shown in Figure a, under the condition of nitrogen level of 0N, the overexpression line OsUBP15OE ( Pro Ubi ::gOsUBP15-GFP ) fluorescence did not accumulate in the root apical meristem and mature zone, but with the increase of nitrogen level, the fluorescence intensity of the overexpression line OsUBP15OE (ProUbi::gOsUBP15-GFP) gradually accumulated in the quiescent central cells of the root apex, and in the cells of the mature zone, the overexpression line OsUBP15OE ( Pro Ubi ::gOsUBP15-GFP ) also accumulated significantly under the condition of 1N. Pro Ubi ::gOsUBP15-GFP The grayscale analysis of OsUBP15-GFP fluorescence intensity in the quiescent central cells of the root tips was performed (Image J software). Picture 6 As shown in Figure b, it can be seen that as the nitrogen level increases, the fluorescence intensity gradually increases, and there is a significant positive correlation between the two. These results prove that the OsUBP15 protein level changes adaptively with the nitrogen level. OsUBP15 Genes positively regulate nitrogen absorption and utilization efficiency, so OsUBP15 The gene is likely to be the key signal node for rice to adaptively regulate nitrogen absorption and utilization in response to external nitrogen levels.

[0084] In summary, OsUBP15 protein is a protein that synchronizes the external nitrogen level and nitrogen absorption rate in rice. The molecular mechanism of rice regulating nitrogen absorption efficiency in response to external nitrogen levels has been preliminarily analyzed: after sensing the external nitrogen level, rice adjusts the OsUBP15 protein level to determine the nitrogen absorption rate, thereby ensuring the growth of plants under different nitrogen levels. This is a new molecular mechanism for plants to respond to external nitrogen levels and coordinate nitrogen absorption and utilization efficiency.

[0085] The Uniprots accession number of OsUBP15 protein is Q0E2F9, and the RefSeq_Locus accession number is XP_015627057; OsUBP15 The gene MSU_Locus number is LOC_Os02g14730, the NCBI_Locus number is LOC4328857, and the RAP_Locus number is Os02g0244300;

[0086] In the process of rice seed improvement, knockout OsUBP15 Genes that significantly reduce rice's sensitivity to nitrogen at different growth stages, thereby reducing nitrogen utilization efficiency;

[0087] in, OsUBP15 Gene knockout is performed using any of the following techniques:

[0088] Knockout using T-DNA insertion OsUBP15 Gene;

[0089] Knockout using gene editing OsUBP15 Gene;

[0090] Knockout by chemical mutagenesis OsUBP15 Gene;

[0091] Knockout by physical mutagenesis OsUBP15 Gene.

[0092] In the process of rice seed improvement, overexpression can also be OsUBP15The gene may enhance the activity of OsUBP15 protein to improve nitrogen utilization efficiency of rice;

[0093] Among them, overexpression OsUBP15 Genes or methods for increasing the activity of OsUBP15 protein include any of the following:

[0094] Overexpression through a strong promoter OsUBP15 Gene;

[0095] Enhanced by mutations in promoter, UTR and other regions OsUBP15 Gene expression;

[0096] The activity of OsUBP15 protein was enhanced by gene mutation.

[0097] In the rice planting process, you can first plant OsUBP15 Gene overexpression strains or strains with enhanced OsUBP15 protein activity can regulate rice growth and improve nitrogen utilization efficiency by regulating nitrogen levels in the planting environment.

[0098] get OsUBP15 The gene overexpression strain or the strain with enhanced OsUBP15 protein activity includes any of the following:

[0099] Overexpression through a strong promoter OsUBP15 Genes to obtain OsUBP15 Gene overexpression strains;

[0100] Enhanced by mutations in promoter and UTR regions OsUBP15 Gene expression to obtain OsUBP15 Gene overexpression strains;

[0101] The activity of OsUBP15 protein is enhanced by gene mutation to obtain a strain with enhanced OsUBP15 protein activity.

[0102] Although the above embodiments have been described in detail, they are only a part of the embodiments of the present invention, rather than all the embodiments. A person skilled in the art can also obtain other embodiments based on the embodiments without inventiveness, and these embodiments all belong to the protection scope of the present invention.

Claims

1. An application of a protein for regulating nitrogen utilization rate of rice, characterized in that: The application is that OsUBP15 protein synchronizes the external nitrogen level and nitrogen absorption rate; The OsUBP15 protein positively regulates the nitrogen uptake rate; The application is to enhance the activity of OsUBP15 protein to improve the nitrogen utilization efficiency of rice; The gene encoding the OsUBP15 protein is OsUBP15 gene, MSU_Locus accession number is LOC_Os02g14730.

2. The use according to claim 1, characterized in that: The protein is a deubiquitinating enzyme OsUBP15 protein that responds to nitrogen and positively regulates nitrogen utilization throughout the growth cycle; OsUBP15 protein is a rice protein that synchronizes external nitrogen levels with nitrogen absorption rate.

3. The use according to claim 1 or 2, characterized in that: The nitrogen utilization rate is positively regulated by nitrogen throughout the growth cycle, including at least one of the following: At the seedling stage, nitrogen promotes the increase of aboveground dry weight; During the vegetative growth period, nitrogen promotes plant growth and increases photosynthetic rate; During the reproductive growth period, nitrogen promotes seed enlargement; Nitrogen promotes the accumulation of OsUBP15 protein.

4. The use according to claim 1 or 2, characterized in that: Said OsUBP15 Genes positively regulate nitrogen utilization efficiency.

5. The use according to claim 4, characterized in that: The application is overexpression OsUBP15 Gene to improve nitrogen use efficiency in rice.

6. The use according to claim 5, characterized in that: The overexpression OsUBP15 Genes or methods for increasing the activity of OsUBP15 protein include any of the following: Overexpression through a strong promoter OsUBP15 Gene; Enhanced by mutations in promoter and UTR regions OsUBP15 Gene expression; The activity of OsUBP15 protein was enhanced by gene mutation.

7. A method for regulating nitrogen utilization efficiency of rice, characterized in that: The method is to plant OsUBP15 Gene overexpression lines or lines with enhanced OsUBP15 protein activity to regulate rice growth; The gene encoding the OsUBP15 protein is OsUBP15 Gene, OsUBP15 The gene MSU_Locus is numbered LOC_Os02g14730.

8. The method according to claim 7, characterized in that get OsUBP15 The gene overexpression strain or the strain with enhanced OsUBP15 protein activity includes any of the following: Overexpression through a strong promoter OsUBP15 Genes to obtain OsUBP15 Gene overexpression strains; Enhanced by mutations in promoter and UTR regions OsUBP15 Gene expression to obtain OsUBP15 Gene overexpression strains; The activity of OsUBP15 protein is enhanced by gene mutation to obtain a strain with enhanced OsUBP15 protein activity.

9. The method according to claim 7 or 8, characterized in that: The method is to plant OsUBP15 In the process of gene overexpression strains or strains with enhanced OsUBP15 protein activity, nitrogen utilization efficiency is improved.

Images