Application of indica rice osrnr10 gene

Abstract

This invention discloses the application of the OsRNR10 gene in indica rice. The OsRNR10 gene is used to enhance root development and improve nitrogen fertilizer use efficiency and yield in rice. The sequence of the OsRNR10 gene is shown in SEQ ID NO.1 and SEQ ID NO.2. We performed QTL analysis on 82 lines carrying the IRAT261 single-segment substitution in the Huajingxian 74 background and further finely mapped a nitrogen-responsive root development regulator, OsRNR10. The haplotype OsRNR10 in indica rice leads to a lower expression level, resulting in better root structure and a higher nitrate nitrogen uptake rate in indica rice. By introducing the superior allelic variation of OsRNR10 from indica rice into japonica rice, root development and nitrogen fertilizer use efficiency and yield in japonica rice can be enhanced.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology. Specifically, this invention relates to the application of OsRNR10 in regulating rice root development, nitrogen fertilizer use efficiency, and yield, as well as its alleles in indica rice. Background Technology

[0002] Rice (Oryza sativa L.) is one of my country's most important food crops, ranking second in the country in terms of planting area and yield. Therefore, green and safe rice production plays a vital role in my country's food security.

[0003] Data from the Food and Agriculture Organization of the United Nations (FAO) shows that global nitrogen fertilizer consumption has increased significantly over the past decade, while food production growth has been slow. Excessive nitrogen fertilizer input has not only failed to substantially increase yields but has also led to a decline in both economic and ecological benefits. How to sustainably increase crop yields while reducing fertilizer inputs has become a critical issue that urgently needs to be addressed to ensure food security and sustainable agricultural development.

[0004] The plastic development of plant roots is mainly influenced by both internal hormonal signals and external environmental factors. Plant hormones play a crucial regulatory role in the early establishment of roots and post-embryonic growth. The combined regulation of multiple hormones endows plant roots with flexibility and plasticity in growth and development, among which auxin plays a vital role in root plasticity (Roychoudhry et al., 2022). To maintain plant growth and development, plant roots need to absorb 14 essential mineral elements from the soil, with nitrogen being one of the most important nutrients. The level of external nitrogen sources is often a significant factor limiting plant root development. Nitrogen-use efficiency (NUE) is a highly complex trait, largely dependent on nitrogen uptake efficiency (NUpE) (Masclaux-Daubresse et al., 2022). Since roots are the primary nitrogen-absorbing organs, NUpE is influenced by root structure. External nitrogen sources greatly influence the root system architecture (RSA) of plants. Generally speaking, low nitrogen conditions lead to stronger RSA, ensuring that plants can obtain external nitrogen sources to the maximum extent under low nitrogen conditions (Jia et al., 2022).

[0005] Current understanding of the synergistic regulation of plant growth and development by nitrogen and auxin mainly stems from research on the model plant Arabidopsis thaliana, and largely focuses on the regulation of RSA (Resin-Free Syndrome). On the one hand, external nitrogen sources can alter the root architecture of Arabidopsis thaliana by regulating the expression of the auxin synthesis gene TAR2 (Tryptophan Aminotransferase Related 2) and the auxin receptor gene AtAFB3 (Auxin-signaling F-box Protein 3) (Ma et al., 2014; Vidalet et al., 2010); on the other hand, low concentrations of nitrate nitrogen (NO3) can also influence the growth and development of plants. - Under certain conditions, the nitrate nitrogen transporter NRT1.1 transports auxin towards the base, leading to a decrease in auxin accumulation in the root tips of lateral roots, thereby inhibiting lateral root growth (Krouk et al., 2010). However, the genetic mechanisms of the auxin pathway differ significantly between monocots and dicots (Wang et al., 2018); how nitrogen and auxin interact to affect rice root development and thus synergistically regulate nitrogen fertilizer use efficiency and yield remains unclear. Summary of the Invention

[0006] The purpose of this invention is to address the aforementioned shortcomings of the prior art by providing an application of the OsRNR10 gene in indica rice.

[0007] Another object of the present invention is to provide a marker relating rice root plasticity development and rice nitrogen fertilizer utilization efficiency.

[0008] Another objective of this invention is to provide a method for improving rice root development and nitrogen fertilizer utilization efficiency.

[0009] The objective of this invention can be achieved through the following technical solutions:

[0010] The application of the indica rice OsRNR10 gene in optimizing rice root development and improving nitrogen fertilizer utilization efficiency and yield in rice. The promoter sequence of the indica rice OsRNR10 gene is shown in SEQ ID NO.1, the gDNA nucleotide sequence is shown in SEQ ID NO.2, the cDNA nucleotide sequence is shown in SEQ ID NO.3, and the encoded protein amino acid sequence is shown in SEQ ID NO.4.

[0011] The promoter sequence of the OsRNR10 gene in japonica rice IRAT261 is shown in SEQ ID NO.5, the gDNA nucleotide sequence is shown in SEQ ID NO.6, the cDNA nucleotide sequence is shown in SEQ ID NO.7, and the encoded protein amino acid sequence is shown in SEQ ID NO.8.

[0012] Application of the OsRNR10 gene promoter in indica rice in optimizing rice root development and improving nitrogen fertilizer utilization efficiency and yield in rice. The OsRNR10 gene promoter sequence of indica rice is shown in SEQ ID NO.1.

[0013] A nitrogen-dependent marker related to rice root development and nitrogen fertilizer use efficiency was identified as a 3496 bp sequence (912–4408 bp) in the promoter of the OsRNR10 gene, as shown in SEQ ID NO. 5. The presence of this marker in japonica rice resulted in high OsRNR10 expression, weakened root development, insensitivity to changes in external nitrogen sources, and a slower NO3- uptake rate. The absence of this marker in indica rice resulted in low OsRNR10 expression, more developed roots, greater sensitivity to changes in external nitrogen sources, and an increased NO3- uptake rate.

[0014] A method for determining root development and nitrogen fertilizer utilization efficiency in rice involves detecting the promoter of the rice OsRNR10 gene. If the marker described in this invention is present, the rice variety has relatively weak root development and low nitrogen fertilizer utilization efficiency; if the marker described in this invention is missing, the rice variety has relatively strong root development and high nitrogen fertilizer utilization efficiency.

[0015] As a preferred embodiment of the present invention, the PCR primers used for detecting the promoter of the rice OsRNR1O gene are shown in SEQ ID NO.9 and SEQ ID NO.10.

[0016] A method for enhancing root development and nitrogen fertilizer use efficiency in rice involves knocking out or silencing the OsRNR10 gene in rice, preferably japonica rice. Using this method, nitrogen fertilizer use efficiency and yield in rice can be improved by optimizing the root structure.

[0017] OsRNR10 promotes the stability of auxin synthesis inhibitor OsDNR1 (DULL NITROGENRESPONSE1) by monoubiquitination modification of the K53 site, thereby inhibiting auxin accumulation, root growth, and NO3- uptake.

[0018] Beneficial effects:

[0019] This invention utilizes 82 single-fragment substitution system materials in the background of Huajingxian 74 (HJX74) ( C hromosome S egment S ubstitution LA comparative analysis of root size under different nitrogen concentration treatments was conducted using the CSSL (Central Society of Lyceum, Ines). Combined with map-based cloning, a nitrogen-responsive root development regulator, OsRNR10 (REGULATOROF N-RESPONSIVE RSA ON CHROMOSOME 10), was located on chromosome 10. Further investigation revealed that OsRNR10 promotes the stability of the auxin synthesis inhibitor OsDNR1 (DULL NITROGEN RESPONSE1) by monoubiquitinizing the K53 site, thereby inhibiting auxin accumulation and suppressing root growth and NO3-. - The absorption of nitrogen fertilizer by OsRNR10 was improved. Knocking out OsRNR10 in japonica rice, reducing its expression level, optimized root structure and improved nitrogen fertilizer use efficiency and yield. This invention further discovered a 3496bp difference in the OsRNR10 promoter region between indica and japonica rice. Specifically, japonica rice has a 3496bp sequence from -3645 to -150bp in its OsRNR10 gene promoter, while indica rice lacks this sequence. In indica rice, this 3496bp deletion leads to reduced OsRNR10 gene expression, resulting in superior root structure and nitrogen fertilizer use efficiency. Therefore, this 3496bp deletion can serve as a marker for assessing root development and nitrogen fertilizer use efficiency in rice. Attached Figure Description

[0020] Figure 1 The root architecture of different indica and japonica materials under different nitrogen culture conditions is shown. (a) Root phenotypes of different indica and japonica subspecies under low nitrogen and high nitrogen culture conditions, (b) Total root length ratio, (c) Total root area ratio, (d) Total root tip number ratio.

[0021] In the diagram, HJX74 is the abbreviation for Huajingxian 74; TQ is the abbreviation for Teqing; ZF802 is the abbreviation for Zhefu 802; ZH11 is the abbreviation for Zhonghua 11; and WYJ7 is the abbreviation for Wuyunjing 7.

[0022] Figure 2 Map-based clones of candidate gene OsRNR10 and their sequence differences between HJX74 and IRAT261. (a) Fine localization of OsRNR10; (b) Differences between the OsRNR10 promoter, open reading frame, and protein-coding region in HJX74 and IRAT261. Figure 3 The expression of OsRNR10 is induced by nitrogen and the OsRNR10 promoter activity is analyzed. (a) The transcriptional level and protein abundance of OsRNR10 increase with increasing external nitrogen concentration. (b) The transcriptional activation assay is used to verify the OsRNR10 promoter activity derived from HJX74 and IRAT261.

[0023] Figure 4 This shows the root phenotype and NO3 content of transgenic materials with OsRNR10 under the ZH11 background. - Absorption rate. (a) Root phenotypes of OsRNR10 transgenic material under low-nitrogen and high-nitrogen culture conditions, (b) Total root length ratio, (c) Total root area ratio, (d) Total root tip number ratio, (e) NO3 - Absorption rate.

[0024] Figure 5 The results show that OsRNR10 and OsDNR1 have protein interactions and affect the stability of OsDNR1. (a) The splitting firefly luciferase complementation assay (SFLC) verified the protein interaction between OsRNR10 and OsDNR1 in plants. (b) The rice protoplast system co-immunoprecipitation (Co-IP) experiment verified the protein interaction between OsRNR10 and OsDNR1. (c) The transcriptional level of OsDNR1 in OsRNR10 transgenic materials. (d) The protein abundance of OsDNR1 in OsRNR10 transgenic materials. (e) The degradation rate analysis of GST-OsDNR1 in OsRNR10 overexpression materials. (f) The degradation rate analysis of GST-OsDNR1 in OsRNR10 materials. (g) The content of endogenous indole-3-acetic acid (IAA) in OsRNR10 transgenic materials.

[0025] Figure 6 This shows that OsRNR10 mediates monoubiquitination of OsDNR1. (a) OsRNR10 modifies OsDNR1 via monoubiquitination, and (b) analysis of the degradation rate of GST-OsDNR1 with different mutation types in OsRNR10 overexpression lines.

[0026] Figure 7 Displaying NIL-OsDNR1 HJX74 NIL-OsDNR1 IRAP9 and NIL-OsDNR1 IRAP9 OsDNR1 expression level, root phenotype, and auxin content in osrnr10. (a) Transcriptional level and protein abundance of OsDNR1, (b) Root phenotype under low-nitrogen and high-nitrogen culture conditions, (c) Total root length ratio, (d) Total root area ratio, (e) Total root tip number ratio, (f) Endogenous IAA content.

[0027] Figure 8The OsRNR10 sequence shows differentiation between indica and japonica rice. (a) Promoter region analysis of OsDNR1 in 12 indica and 12 japonica rice varieties, (b) Open reading frame region analysis, (c) Protein coding region analysis, (d) Transcriptional level of OsDNR1, (e) Phylogenetic tree of OsRNR10 in approximately 3000 rice varieties, (f) Haplotype analysis of OsRNR10, (g) Nucleotide diversity analysis in the upstream and downstream 20kb regions of OsRNR10, and (h) Haplotype distribution of OsRNR10 and OsDNR1.

[0028] Figure 9 Showing near-isogenic line NIL-OsRNR10 HJX74 and NIL-OsRNR10 IRAT261 Root system changes and major agronomic traits under different nitrogen concentrations. (a) Total root length ratio under low nitrogen and high nitrogen culture conditions, (b) Total root area ratio, (c) Total root tip number ratio, (d) Plant height, (e) Tillering, (f) Number of secondary branches, (g) Number of grains per ear, (h) Yield per plant.

[0029] Figure 10 The phenotypic analysis of important agronomic traits of ZH11 and its background knockout line osrnr10 under different nitrogen application levels is shown. (a) Plant type, (b) Plant height, (c) Tillering, (d) Number of primary branches, (e) Number of secondary branches, (f) Number of grains per panicle, (g) Yield per plant. Detailed Implementation

[0030] In the following examples, HJX74 is an abbreviation for Huajingxian 74, and ZH11 is an abbreviation for Zhonghua 11.

[0031] Example 1: Confirmed significant differences in the overall size of the RSA and nitrogen-dependent plasticity developmental response between indica and japonica rice.

[0032] The inventors hydroponically cultured four indica rice samples and four japonica rice samples for 10 days in two nutrient solutions: high nitrogen (1.25 mM NH4NO3) and low nitrogen (0.375 mM NH4NO3). Root development indices (root length, root area, and number of root tips) were then measured under both high and low nitrogen conditions, and the ratios of these indices to those under high and low nitrogen conditions were calculated. The results showed that, compared to indica rice, japonica rice had a lower overall RSA (Radioactive Standard Amount) and a weaker response to external nitrogen. Figure 1 ).

[0033] Example 2: Identification of the key site OsRNR10 leading to the difference in RSA nitrogen response between indica and japonica rice

[0034] The inventors constructed 82 SSSLs (Spiritual Strains) materials using IRAT261 as the donor parent and HJX74 as the recipient parent. These were cultured hydroponically, and it was found that SSSL-024 had a smaller overall RSA and a weaker response to nitrogen compared to HJX74. SSSL-024 carries a fragment from IRAT261 on the short arm of chromosome 10. To finely locate root development regulators of nitrogen response, we backcrossed SSSL-024 with HJX74 to construct a population of 106 BC1F2 plants and 621 BC2F2 plants for fine mapping and map-based cloning. Candidate genes were located within a 3.3 kb range between markers P241 and P242. Only one gene, LOC_Os10g41838, exists within this region, and its promoter and genomic region are identified. This gene is named OsRNR10 (REGULATOR OF N-RESPONSIVE RSA ON CHROMOSOME 10). Figure 2 a). Sequencing and sequence alignment revealed that SSSL-024's OsRNR10 has an additional 3496 bp fragment in the promoter region (SEQ ID NO. 5, 912–4408 bp), while HJX74's OsRNR10 has a 604 bp insertion in the open reading frame region (SEQ ID NO. 2, 103–707 bp). However, this fragment was spliced ​​out during transcription and therefore did not affect the protein-coding sequence. Figure 2 b).

[0035] The molecular markers used in this study were PCR-based, including SSR markers and self-designed InDel markers. All SSR markers were derived from the microsatellite marker linkage map published by McCouch et al. (2001, 2002). STS markers were selected by analyzing clone sequences using an SSR analysis tool (http: / / www.gramene.org / gramene / searches / ssrtool) to identify SSR target sequences with good microsatellite reproducibility, and then primers were designed for these target sequences using Primer5 analysis software. The polymorphic marker primer sequences used for fine mapping and map-based cloning are detailed in Table 1.

[0036] Table 1

[0037]

[0038]

[0039] The PCR procedure was slightly modified from the method of Panaud et al. (1996). Specifically, each 20 μL amplification reaction tube contained: 0.15 μM SSR primers, 200 μM dNTPs, 1× PCR reaction buffer (50 mM KCl, 10 mM Tris-HCl pH 8.3, 1.5 mM MgCl2, 0.01% gelatin), 50–100 ng template DNA, and 1 U Taq enzyme. The reaction program was: DNA denaturation at 94℃ for 5 min, followed by 36 cycles of (94℃ 1 min, 56℃ 1 min, 72℃ 1 min) and a final extension at 72℃ for 5 min. The amplified PCR products were electrophoresed on a 6% polyacrylamide denaturing gel, and the gel was imaged after electrophoresis.

[0040] Example 3: OsRNR10 expression increased with increasing nitrogen concentration, and OsRNR10... IRAT261 The promoter activity is higher than that of OsRNR10. HJX74 promoter activity

[0041] The inventors conducted a hydroponic experiment on HJX74 using nutrient solutions with four nitrogen concentration gradients (0.15N, 0.1875mM NH4NO3; 0.3N, 0.375mM NH4NO3; 0.6N, 0.75mM NH4NO3; and 1N, 1.25mM NH4NO3). Total RNA was extracted from the root tips of HJX74 plants after 4 weeks of hydroponics, and the transcriptional and protein levels of OsRNR10 were analyzed using qRT-PCR and Western blotting. The results showed that the expression level of OsRNR10 increased with increasing nitrogen concentration. Figure 3 a). Furthermore, the inventors also found through protoplast transient expression analysis that the OsRNR10 promoter activity from IRAT261 was higher than that from HJX74 ( Figure 3 b).

[0042] The transcriptional activation assay used in this embodiment is briefly described as follows: Approximately 1.5 kb and 5 kb OsRNR10 promoter fragments were amplified from HJX74 and IRAT261, respectively (primer sequences are shown in Table 1, OsRNR10-G0800), and inserted upstream of the LUC gene in the pGreenII0800-LUC vector to construct pHJX74. 1500 ::LUC vector and pIRAT261 5000::LUC carrier. Rice seedling coleoptiles were cut into strips and placed in an enzymatic hydrolysate. After lysis by low-speed shaking in the dark for 5 hours, an equal volume of W5 solution (154 mM NaCl, 125 mM CaCl2, 5 mM KCl, 2 mM MES, pH 5.7) was added. The mixture was centrifuged at 200 g for 5 minutes, the precipitate was collected, and the precipitate was resuspended and washed twice with W5 solution. The precipitate was then resuspended in MMg solution (0.4 M mannitol, 15 mM MgCl2, 4 mM MES, pH 5.7). pGreenII 0800-LUC and pHJX74 were then added. 1500 ::LUC vector and pIRAT261 5000 ::LUC was added to rice protoplasts, using an equal volume of PEG4000 / Ca 2+ The transformation was performed, and after standing for 15 minutes, 2 volumes of W5 were added to terminate the reaction and the cells were washed. The cells were then incubated in the dark in W5 solution for 16 hours. Protoplasts were collected and lysis buffer was added. The LUC / REN ratio was determined using the PROMEGA dual-fluorescence detection kit.

[0043] Example 4: Knockout and overexpression lines were constructed in the context of the japonica rice variety ZH11 to confirm that OsRNR10 regulates RSA and NO3 in rice. - Absorption rate

[0044] To further clarify the function of OsRNR10, the inventors constructed an OsRNR10 knockout vector using a CRISPR / Cas9 knockout system; simultaneously, they constructed an OsRNR10 overexpression vector pAct::OsRNR10-Flag. Using Agrobacterium-mediated transformation, both vectors were introduced into the japonica rice variety ZH11, creating the OsRNR10 knockout line osrnr10 and the overexpression line ZH11 / pAct::OsRNR10-Flag. Subsequently, we used the high-low nitrogen hydroponic system from Example 1 to measure the root development indices of ZH11 / pAct::OsRNR10-Flag and osrnr10. We found that ZH11 / pAct::OsRNR10-Flag and osrnr10 showed less sensitive RSA responses to external nitrogen application compared to wild-type ZH11, with ZH11 / pAct::OsRNR10-Flag exhibiting a smaller RSA, while osrnr10 showed a larger RSA. Figure 4 We also measured its NO3- absorption rate, and the results showed that compared with wild-type ZH11, the NO3- absorption rate of ZH11 / pAct::OsRNR10-Flag was significantly decreased, while the NO3- absorption rate of osrnr10 was significantly increased. Figure 4 e).

[0045] This example uses NO3.- The absorption rate was determined by disinfecting rice seeds with a 20% sodium hypochlorite solution for 30 minutes. Afterward, the seeds were placed in a 37℃ incubator for 24 hours to absorb water and swell. The seeds were then drained and transferred to a 28℃ incubator for germination. Once the seeds showed signs of germination, they were transferred to perforated 96-well plates and cultured for 7 days. Seedlings with uniform growth were then transferred to a 40L nutrient solution containing (1.25mM NH4NO3, 0.5mM NaH2PO4·2H2O, 0.75mM K2SO4, 1mM CaCl2, 1.667mM MgSO4·7H2O, 40μM Fe-EDTA(Na), 19μM H3BO3, 9.1μM MnSO4·H2O, 0.15μM ZnSO4·7H2O, 0.16μM CuSO4, and 0.52μM (NH4)3Mo7O). 24 The solution was placed in a blue box containing 4H2O (pH 5.5). For different nitrogen concentration treatments, standard nutrients were added: 0.3N (0.375mM NH4NO3) and 0.15N (0.1875mM NH4NO3). The solution was cultured for 3 weeks, with the pH adjusted daily.

[0046] After 3 weeks of cultivation, the rice roots were immersed in 0.1 mM CaSO4 for 1 minute, and then transferred to a solution containing 2.5 mM K. 15 Immerse the roots in a nutrient solution containing NO3 for 5 minutes, then transfer to 0.1 mM CaSO4 for 1 minute. Blot the roots dry with filter paper or gauze, cut off the roots, dry them, grind them, and then measure the moisture content. 15 Nitrogen content (computed by Li Yuzhong's laboratory at the Chinese Academy of Agricultural Sciences, using an Isoprime 100 instrument).

[0047] Example 5: OsRNR10 positively regulates the stability of the auxin synthesis inhibitor OsDNR1, thereby reducing auxin accumulation in rice.

[0048] The inventors predicted through protein domain analysis that OsRNR10 belongs to the FBA protein family, which is part of the F-box family, and may function as an F-box protein. Therefore, to determine the downstream targets of OsRNR10, we performed immunoprecipitation combined with mass spectrometry (IP-MS) and identified four high-quality peptides derived from OsDNR1. Furthermore, we further confirmed the interaction between OsRNR10 and OsDNR1 through a split firefly luciferase complementation assay (SFLC) and a rice protoplast immunoprecipitation assay (Co-IP). Figure 5ab). We then examined the transcriptional level and protein abundance of OsDNR1 in ZH11 / pAct::OsRNR10-Flag and osrnr10. The results showed that although no difference was detected in the transcriptional level of OsDNR1, the protein abundance of OsDNR1 was significantly increased in ZH11 / pAct::OsRNR10-Flag, while it was significantly decreased in osrnr10. Figure 5 The result (cd) indicates that OsRNR10 can regulate the protein abundance of OsDNR1 at the protein level.

[0049] Based on this, we used a cell-free degradation assay to detect the degradation rate of GST-OsDNR1 (glutathione thiotransferase) in wild-type ZH11, ZH11 / pAct::OsRNR10-Flag, and osrnr10. The results showed that, compared with wild-type, the degradation rate of GST-OsDNR1 decreased in ZH11 / pAct::OsRNR10-Flag, but increased in osrnr10. Figure 5 Meanwhile, we measured the auxin content of the OsRNR10 transgenic material. The results showed that the IAA level decreased in ZH11 / pAct::OsRNR10-Flag, while more IAA accumulated in osrnr10. Therefore, we speculate that OsRNR10 positively regulates the stability of the auxin synthesis inhibitor OsDNR1, thereby reducing auxin accumulation in rice.

[0050] The SFLC experimental method used in this embodiment is briefly described as follows: Full-length OsRNR10 and OsDNR1 cDNA were amplified (using ZH11 as a template, primers are shown in Table 1, OsRNR10-Getway), and then inserted into pCAMBIA1300-35SCluc-RBS and pCAMBIA3300-35S-HA-Nluc-RB, respectively (Chen, H. et al. Firefly luciferase complementation imaging assay for protein-protein interactions in plants. Plant Physiol. 146, 368-376 (2008).). cLUC-OsRNR10, OsDNR1-Nluc, and the silencing plasmid p19 were simultaneously transfected into tobacco epidermal cells via Agrobacterium-mediated transformation. After 48 h of incubation, the injected tobacco leaves were coated with 1 mM luciferin (Promega, E1605), and the LUC signal was observed using an LB985 (Berthold) microscope. The carrier and specific experimental methods were as described by Chen et al. (2008).

[0051] The Co-IP experimental method used in this embodiment is briefly described as follows: Full-length OsRNR10 and OsDNR1 cDNA were amplified (using ZH11 as a template, primers are shown in Table 1 OsRNR10-Getway), and then inserted into pUC-35S-Flag-RBS and pUC-35AS-HA-RBS vectors, respectively (Liu, Q. et al. G-protein βγ subunits determine grain size through interaction with MADS-domain transcription factors in rice. Nat. Commun. 9, 852 (2018).). Rice protoplasts were transfected with 100 μg of plasmid (rice protoplast preparation method as described in Example 3), cultured under low light for 18 h, and then subjected to 50 mM HEPES (pH 7.5), 150 mM KCl, 1 mM EDTA (pH 8), 0.3% Tritium-X 100, 1 mM DTT, and added protease inhibitors (Roche Life Science). The lysates were incubated with magnetic beads conjugated to DDDDK-tagged antibodies (MBL, M185-11) at 4°C for 4 hours. The beads were then washed six times with lysis buffer and eluted with 3× Flag peptide (Sigma-Aldrich, F4709). Immunoprecipitates were separated by SDS-PAGE electrophoresis, and target proteins were detected by Western blotting using DDDDK (MBL, M185-7) or HA (MBL, M180-7) antibodies. The immunoblotting results were visualized on a Tanon-5200 chemiluminescence imaging system (Tanon Science and Technology). The vectors and specific experimental methods were as described by Liu et al. (2018).

[0052] The cell-free degradation assay method used in this embodiment is briefly described as follows: Rice seedlings grown for 14 days were collected, ground with liquid nitrogen, and cell lysates were extracted using 25 mM Tris-HCl (pH 7.5), 10 mM NaCl, 10 mM MgCl2, 4 mM PMSF, 5 mM DTT, and 10 mM ATP. 200 μL of rice cell lysates were incubated with 100 ng of purified GST-OsDNR1 fusion protein for a series of time periods. Proteins were extracted and separated by SDS-PAGE electrophoresis. Target proteins were detected by Western blotting using an anti-GST antibody (MBL, PM013-7). Ponceau S staining was used as a control.

[0053] Example 6: OsRNR10 inhibits the ubiquitination and degradation of OsDNR1 by monoubiquitination of the K53 site of OsDNR1.

[0054] Mass spectrometry analysis revealed that three lysine sites (K53, K314, and K368) of OsDNR1 are ubiquitinated in plants. Since OsRNR10 may act as an F-box protein in plants to form the SCF complex and function as an E3 ubiquitin ligase to ubiquitinate substrates, we conducted in vitro ubiquitination experiments. The results showed that GST-OsDNR1 could be monoubiquitinated by the OsRNR10-Flag fusion protein in the presence of ubiquitin activator E1, ubiquitin conjugate E2, and ubiquitin. Figure 6 a). To further elucidate the sites of OsRNR10 monoubiquitination of OsDNR1, we examined the degradation rates of mutant fusion proteins of GST-OsDNR1 with one or more lysine residues replaced by alanine in cell-free degradation systems of ZH11 and ZH11 / pAct::OsRNR10-Flag. The results showed that, compared to wild-type GST-OsDNR1, the GST-OsDNR1 mutant with phenylalanine replacing lysine at the K53 site was no longer stable in ZH11 / pAct::OsRNR10-Flag, while mutations altering the lysine residues at the other two sites still improved the stability of GST-OsDNR11 in ZH11 / pAct::OsRNR10-Flag. Figure 6 b). Therefore, we conclude that OsRNR10 inhibits the ubiquitination and degradation of OsDNR1 by monoubiquitination of the K53 site of OsDNR1.

[0055] The in vitro ubiquitination assay used in this embodiment is briefly described as follows: OsRNR10 fusion protein was immunoprecipitated from the protein extract of ZH11 pAct::OsRNR10-Flag using magnetic beads conjugated to a DDDDK-tagged antibody (MBL, M185-11), followed by elution with 3×Flag peptide (Sigma-Aldrich). The purified OsRNR10-Flag protein was used for in vitro ubiquitination assays. GST-OsDNR1, HA-Ubiquitin (U-110-01M, R&D), GST-E1 (UBE1, E-306-050, R&E), E2 (E2-607-100, R&D), and purified E3 (RNR10-Flag) were used for protein ubiquitination analysis. The buffer contained 50 mM Tris-HCl (pH 7.4), 5 mM MgCl2, and 2 mM ATP. The reactants were incubated at 30°C for 5 hours, and the products were separated by SDS-PAGE electrophoresis. The proteins were then detected by immunoblotting using OsDNR1 antibody (ABclonal) and Ubiquitin antibody (abcam, ab134953).

[0056] Example 7: OsRNR10 acts upstream of OsDNR1 to regulate auxin homeostasis and root phenotype.

[0057] The inventors previously constructed a near-isogenic NIL-OsDNR1 of OsDNR1. IRAP9 In this context, the OsRNR10 knockout system NIL-OsDNR1 was constructed using the CRISPR / Cas9 knockout system. IRAP9 osrnr10, then we tested NIL-OsDNR1 HJX74 NIL-OsDNR1 IRAP9 NIL-OsDNR1 IRAP The transcriptional level and protein abundance of OsDNR1 in osrnr10 were analyzed. The results showed that in NIL-OsDNR1... IRAP The transcriptional level of OsDNR1 in osrnr10 remained unchanged, but its protein abundance decreased significantly. Figure 7 a). Root phenotypic analysis also showed NIL-OsDNR1 IRAP9 The root system of osrnr10 is further less sensitive to nitrogen. Figure 7 be), and the level of endogenous auxin was also increased ( Figure 7 f). Based on the above results, we conclude that OsRNR10 acts upstream of OsDNR1, regulating auxin homeostasis and root phenotype in rice.

[0058] Example 8: The differentiation between indica and japonica rice due to OsRNR10 caused differences in the RAS response of indica and japonica rice to external nitrogen.

[0059] With OsRNR10 HJX74 Compared to the promoter of the gene (SEQ ID NO.1), the allele OsRNR10 IRAT261 The promoter (SEQ ID NO.5) contains a 3496 bp insertion fragment and 25 SNPs of difference, while OsRNR10 HJX74 There is a 603 bp insertion in the open reading frame region (SEQ ID NO.2). The inventors analyzed the OsRNR10 gene in 12 indica and 12 japonica rice materials (materials shown in Table 2) and found that all indica rice varieties had a 3496 bp deletion in the promoter region and a 604 bp insertion in the open reading frame region. Furthermore, the 604 bp insertion in the open reading frame region was spliced ​​out during transcription and did not affect the encoded amino acid sequence. Figure 8 Meanwhile, we detected the OsRNR10 transcription level in these 24 materials and found that its transcription level in japonica rice varieties was significantly higher than that in indica rice varieties. Figure 8d). Therefore, the deletion of 3496 bp in the OsRNR10 promoter region in indica rice varieties leads to low OsRNR10 expression, resulting in a higher overall RSA and stronger response to external nitrogen in indica rice, and NO3. - The absorption rate is also higher.

[0060] The inventors further conducted phylogenetic analysis using approximately 3,000 rice germplasm accessions, and the results showed that OsRNR10 exhibits significant differentiation in both indica and japonica rice. Figure 8 e) Haplotype analysis of these varieties showed that OsRNR10 has four haplotypes, with Hap.I and Hap.II rice varieties accounting for the majority (83.9%), and 98.7% of japonica rice being Hap.II and 69.1% of indica rice being Hap.I, demonstrating a strong preference for indica-japonica subspecies selection. Figure 8 f). Simultaneously, nucleotide polymorphism analysis of the upstream and downstream 20kb of OsRNR10 also demonstrated that directional selection drives the differentiation of OsRNR10 in indica and japonica rice. Figure 8 g). Haplotype distribution pattern analysis of OsRNR10 and OsDNR1 showed a large degree of overlap, indicating that these two genes synergistically regulate RAS, nitrogen response, and NO3 in rice. - Absorption rate ( Figure 8 h).

[0061] Table 2

[0062] Rice variety name Subspecies Xiang Ai Zao No. 4 Indica rice Guanglu Dwarf No. 4 Indica rice Minghui 63 Indica rice Wen Xuanqing Indica rice Yuan Fengzao Indica rice Xiang Ai Zao No. 9 Indica rice Early selection of youth Indica rice Zhejiang Fu 802 Indica rice Special Youth Indica rice 9311 Indica rice Nanjing No. 6 Indica rice Huajingxian 74 Indica rice 313 Japonica rice 314 Japonica rice Chinese flower 11 Japonica rice Wuyunjing No. 7 Japonica rice Taichung 65 Japonica rice Xiushui09 Japonica rice Xiushui 110 Japonica rice Nippon Haru Japonica rice Longdao No. 5 Japonica rice Lansheng Japonica rice Thousand Waves No. 2 Japonica rice

[0063] Example 9: Construction of near-isogenic line NIL-OsRNR10 in the background of indica rice variety HJX74 HJX74 and NIL-OsRNR10 IRAT261 And complete the comparative analysis of agronomic traits such as yield.

[0064] The inventors selected individuals carrying OsRNR10 from the BC2F2 population described in Example 2. IRAT261 The material from the locus was backcrossed three times with HJX74, ultimately yielding a near-isogenic line NIL-OsRNR10 under the HJX74 background. HJX74 and NIL-OsRNR10 IRAT261 Near-isogenic lines were hydroponically cultured under high-nitrogen and low-nitrogen conditions, and root development indices were measured. Results showed that, compared with NIL-OsRNR10... HJX74 Compared to NIL-OsRNR10 IRAT261 The overall RSA is smaller and the response to external nitrogen is weaker. Figure 9(ac). Yield trials were conducted in a field with normal nitrogen application (210 kg / ha), and various important agronomic traits were observed and statistically analyzed. Comparative statistical results showed that the allelic variation OsRNR10 in japonica rice... IRAT261 It increased the number of rice tillers, but significantly reduced plant height, the number of primary branches, the number of secondary branches, and the number of grains per panicle, ultimately leading to a decrease in yield per plant. Figure 9 dh).

[0065] Specific statistical methods: Plant height statistics: After rice matures, plant height is measured on 16 plants selected from the field. Tiller statistics: After rice matures, tiller number is measured on 12 plants selected from the field. Grain count per panicle statistics: After rice matures, panicles on the main tillers of 12 plants are collected from the field, and the number of grains per panicle is counted and recorded. Yield per plant statistics: After rice is fully mature, threshing is performed on 12 individual plants in the plot. The harvested seeds are dried at a constant temperature of 37℃ and then weighed to obtain yield data per plant. Three replicate experiments are required.

[0066] Example 10: Performance of agronomic traits such as yield in OsRNR10 knockout lines under different nitrogen application levels in japonica rice ZH11 background

[0067] Field trials were conducted using the OsRNR10 knockout line osrnr10 constructed by the inventors in Example 4. Wild-type ZH11 and the knockout line osrnr10 were planted in fields with different nitrogen application rates (60 kg / ha, 120 kg / ha, 210 kg / ha, and 300 kg / ha), and the agronomic traits of the knockout line and wild-type were observed and statistically analyzed. The results showed that, at the same nitrogen application rate, osrnr10 slightly increased rice plant height, decreased tiller number, and significantly increased the number of secondary branches and grains per panicle. Therefore, compared to ZH11, osrnr10 exhibited higher nitrogen fertilizer use efficiency and higher yield per plant. Furthermore, under low nitrogen fertilizer conditions, the yield increase of osrnr10 was more significant. Figure 10 This indicates that reducing the expression of OsRNR10 can enable rice to maintain a relatively good yield even under low nitrogen application levels, thus saving on nitrogen fertilizer application.

Claims

1. OsRNR10 Application of the gene in optimizing the root system development of indica rice and improving the nitrogen fertilizer use efficiency and yield of rice OsRNR10 The gene promoter sequence is shown as SEQ ID NO. 1, and the gDNA nucleotide sequence is shown as SEQ ID NO.

2.

2. A nitrogen-dependent marker related to rice root development and nitrogen fertilizer use efficiency, characterized in that... The marker is shown as SEQ ID NO. 5 OsRNR10 a 3496 bp sequence from 912~4408 bp in the gene promoter; the marker exists in japonica rice, leading to OsRNR10 high expression, weak development of root system, insensitivity to changes of external nitrogen source, and slow absorption rate of nitrate nitrogen; the marker is deleted in indica rice, leading to OsRNR10 low expression, more developed root system, more sensitivity to changes of external nitrogen source, and increased absorption rate of nitrate nitrogen.

3. A method for determining root development and nitrogen fertilizer utilization efficiency in rice, characterized in that... Detecting rice OsRNR10 a gene promoter, if the marker described in claim 2 is present, the rice variety has relatively weak root development and low nitrogen use efficiency; If the marker described in claim 2 is missing, the rice variety has relatively strong root development and high nitrogen fertilizer utilization efficiency.

4. The method according to claim 3, characterized in that... Testing rice OsRNR1O The PCR primers used for the gene promoter are shown in SEQ ID NO.9 and SEQ ID NO.

10.

5. A method for enhancing root development and nitrogen fertilizer utilization efficiency in rice, characterized in that, Knockout or silence in japonica rice OsRNR10 Genes, the japonica rice mentioned OsRNR10 The gene promoter sequence is shown in SEQ ID NO.5, and the gDNA nucleotide sequence is shown in SEQ ID NO.6.

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