Application of rice OsRNU9 gene
By regulating the expression of the rice OsRNU9 gene, the problem of low nitrogen fertilizer utilization efficiency in rice has been solved, achieving more efficient nitrogen fertilizer utilization and yield improvement, and supporting green and high-yield sustainable development.
Patent Information
- Application Number
- CN202511284937.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-09
- Publication Date
- 2026-01-20
AI Technical Summary
Existing rice varieties have low nitrogen fertilizer utilization efficiency, which means that high yields depend on a large input of nitrogen fertilizer, making it difficult to achieve the sustainable development goal of green and high yield.
Nitrogen fertilizer use efficiency can be regulated by knocking out or increasing the expression of the rice OsRNU9 gene. The deletion of the OsRNU9 gene leads to a decrease in nitrogen fertilizer use efficiency, while increasing its expression can improve nitrogen fertilizer use efficiency and yield.
It improved nitrogen fertilizer utilization efficiency and yield in rice, reduced dependence on nitrogen fertilizer, and achieved more efficient nitrogen fertilizer utilization and yield increase.
Smart Images
Figure CN121362762A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The application belongs to the technical field of plant genetic engineering, and discloses an application of OsRNU9 in regulating nitrogen fertilizer utilization efficiency and yield of rice. BACKGROUND
[0002] Rice (Oryza sativa L.) is one of the most important food crops in the world and is the staple food of more than half of the world's population. Therefore, the safe and efficient production of rice is of great importance to the world's food security. At present, the global population is growing rapidly. According to the Population Division of the Department of Economic and Social Affairs of the United Nations, the world population will grow to 10.9 billion by 2100. The 2024 World Food Security and Nutrition Report points out that by the end of 2030, there will still be 582 million people in the world who will be in a long-term food shortage, making the prospect of achieving the world's ambitious goal of "zero hunger" even more bleak. Achieving the sustainable development goal of a world with food security is the common responsibility of all countries in the world. As an important participant in the international arena, China shoulders an even greater responsibility and must uphold the sense of community of shared destiny and show great power. In order to improve the yield of major cereal crops in the world, it is of great practical significance to study the molecular mechanisms of improving rice yield in the theory of rice breeding.
[0003] In the early 1960s, the successful cultivation of semi-dwarf wheat and rice varieties solved the contradiction between high yield and lodging, improved the harvest index, and greatly increased the yield of crops, greatly alleviating the food crisis caused by rapid population growth. It is known as the first "green revolution". The "green revolution" varieties inhibit the metabolism or signal transduction pathway of gibberellin (Gibberellin Acid, GA), endowing crops with semi-dwarf characteristics, greatly improving the nitrogen tolerance and harvest index of crops.
[0004] However, this also leads to a decrease in the nitrogen uptake capacity of the root system of semi-dwarf varieties and a decrease in the response to nitrogen, resulting in a decrease in nitrogen use efficiency (Nitrogen Use Efficiency, NUE). Therefore, although the yield of semi-dwarf varieties is high, it depends on the large amount of nitrogen fertilizer input. Since the 1970s, China's grain yield has shown a trend of continuous growth, but this is mainly due to the continuous increase in the amount of nitrogen fertilizer applied.
[0005] Although a number of genes that can improve rice NUE have been identified, there is still a long way to go to achieve truly "green, high-yielding" rice varieties. New genetic resources need to be continuously explored, and the regulatory network in plant development and nutrient metabolism needs to be analyzed and identified to provide gene resources and germplasm resources for green, high-yield and efficient breeding. SUMMARY
[0006] The application aims at solving the above problems in the prior art and provides the application of the rice OsRNU9 gene.
[0007] Another object of the application is to provide a potential method for improving the nitrogen fertilizer utilization efficiency of rice.
[0008] The object of the application can be achieved by the following technical solutions.
[0009] The rice OsRNU9 gene has a nucleotide sequence as shown in SEQ ID NO. 1.
[0010] The gene is the OsRNU9 gene shown in SEQ ID NO. 1, and the deletion of the gene will result in a decrease in the expression level of the nitrogen metabolism-related gene in rice, and ultimately result in a decrease in the nitrogen fertilizer efficiency.
[0011] A potential method for improving the nitrogen fertilizer utilization efficiency of rice. Knocking out OsRNU9 will result in a decrease in the nitrogen fertilizer utilization efficiency of rice, and vice versa, increasing the expression of OsRNU9 can improve the nitrogen fertilizer utilization efficiency and yield of rice.
[0012] Beneficial effects:
[0013] The wild-type material WYJ7 is subjected to high and low nitrogen treatment, and the RNA-seq experiment finds that the OsRNU9 gene in rice shows a response to the external nitrogen supply level, and the fluorescence quantitative PCR proves that OsRNU9 has a significant tendency to be induced by low nitrogen. Therefore, the mutant material osrnu9 is constructed, and the 15N isotope labeled nitrogen absorption rate determination and field phenotype investigation of the wild-type material and the mutant material find that knocking out the OsRNU9 gene will reduce the nitrate nitrogen absorption rate, tillering, plant height and yield of rice, and the function of the OsRNU9 gene in positively regulating the nitrogen fertilizer utilization efficiency of rice is determined, and a technical route for improving the nitrogen fertilizer utilization efficiency of rice by increasing the expression of OsRNU9 in rice is proposed. BRIEF DESCRIPTION OF DRAWINGS
[0014] Figure 1 The transcript abundance of OsRNU9 under different nitrogen concentrations is shown.
[0015] Figure 2 The mutation type of the OsRNU9 gene of the mutant material osrnu9 is shown. The structure of the OsRNU9 gene is shown in the figure, the exons are represented by black boxes, the introns are represented by lines, and the nucleic acid sequence below the gene structure is the target sequence.
[0016] Figure 3Phenotype comparison of important agronomic traits between wild type WYJ7 and mutant material osrnu9. (a), field phenotype of osrnu9 mutant material; (b), plant height; (c), tiller; (d), yield per plant; (e), nitrogen uptake rate. DETAILED DESCRIPTION
[0017] WYJ7 in the following examples is the abbreviation of Wu Yunj 7.
[0018] Example 1: OsRNU9 responds to changes in external nitrogen supply level
[0019] To preliminarily verify whether OsRNU9 gene responds to the external nitrogen supply level, the japonica rice material WYJ7 cultured under four nitrogen concentrations (0.15N, 0.1875mM NH4NO3; 0.3N, 0.375mM NH4NO3; 0.6N, 0.75mM NH4NO3; 1N, 1.25mM NH4NO3) was used to extract total RNA for reverse transcription, and then the transcription level of OsRNU9 was detected by fluorescence quantitative PCR, and whether the expression amount changes with the change of external N concentration was analyzed. The specific detection method is as follows: total RNA was extracted from different plant tissues using TRIzol reagent, and full-length cDNA was reverse transcribed using cDNA synthesis kit (TransGen, AT341). Then RT-PCR reaction was carried out according to the instructions (TransGen, AQ601), and the reaction conditions were as follows: first step, 94℃ pre-denaturation for 3min; second step, 98℃ denaturation for 15s; third step, 58℃ annealing for 15s; fourth step, 72℃ extension for 20s; second step, third step and fourth step were executed for 45 cycles; fifth step, melting curve analysis. Each RT-qPCR detection included at least three biological replicates. Rice ACTIN1 gene (OsActinl, LOC_Os03g50885) was used as an internal reference. The relevant RT-qPCR primer sequences are shown in Table 1.
[0020] Table 1
[0021]
[0022] The results show that OsRNU9 responds to the external nitrogen supply level, and the expression level is induced by low concentration of nitrogen Figure 1
[0023] Example 2: Construction of rice OsRNU9 gene mutant osrnu9
[0024] To verify that OsRNU9 is a key site that can regulate the nitrogen uptake rate of rice, we used the CRISPR-Cas9 system to construct the knockout material of OsRNU9. First, the target sequence of OsRNU9 was designed through the CRISPR-P website, and the PCR product was amplified with the template of the rice U6+U3 promoter transcription unit (SEQ ID NO. 2). The PCR product was gel recovered and ligated with the TKC vector which had been completely digested by Spe I (Yubing He; Min Zhu; Lihao Wang; Qiaoyan Wang; Rongchen Wang; Yunde Zhao; Improvements of TKC Technology Accelerate Isolation of Transgene-Free CRISPR / Cas9-Edited Rice Plants. Rice Science; 2019, 26(2): 109-117). Finally, the positive transformants were obtained after transforming E. coli and sequencing. The constructed OsRNU9 knockout vector was transformed into Agrobacterium by Agrobacterium transformation method, and the target gene was inserted into the genome of Agrobacterium. With the help of Agrobacterium infection of plants, the transfer and integration of foreign genes into plant cells were realized. The OsRNU9 gene mutant osrnu9 was obtained by introducing it into japonica rice WYJ7. The primer sequences for constructing the osrnu9 knockout vector are shown in Table 2.
[0025] Table 2
[0026]
[0027] Sequencing analysis found that OsRNU9 inserted a base C at the first exon, which ultimately caused premature termination ( Figure 2 ) of the protein.
[0028] Example 3: Comparison and analysis of nitrogen uptake rate and yield of mutant osrnu9 and other agronomic traits
[0029] In the field yield test, wild type WYJ7 and mutant material osrnu9 were planted in the field (the amount of nitrogen applied was 210 kg / ha), and various important agronomic traits were observed and counted.
[0030] Specific statistical methods: The plant height was measured by taking 16 plants in the field after the rice matured. The number of grains per panicle was counted by taking 16 panicles on the main tillers in the field after the rice matured, and the number of grains per panicle was directly counted and recorded. The yield per plant was obtained by taking 16 single plants in the plot after the rice was completely matured, drying the harvested seeds at 37°C, and weighing the single plant yield data. Three repeated tests were required.
[0031] Statistical results showed that the mutant osrnu9 had reduced plant height and tillering, weakened plant growth, and ultimately reduced yield per plant. Figure 3 ad).
[0032] Subsequently, we studied the wild-type WYJ7 and the mutant material osrnu9. 15 Nitrogen uptake rate was measured. The specific method for determining nitrogen uptake rate was as follows: Seeds of WYJ7 and OsRNU9 were disinfected with a 20% sodium hypochlorite solution for 30 minutes. Afterward, they were placed in a 37℃ incubator for 24 hours to absorb water and allow to swell. The water was then drained, and the seeds were 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 selected and transferred to a nutrient solution containing 40 L of nutrient solution (1.25 mM NH4NO3, 0.5 mM NaH2PO4·2H2O, 0.75 mM K2SO4, 1 mM CaCl2, 1.667 mM MgSO4·7H2O, 40 μM Fe-EDTA(Na), 19 μM M3BO3, 9.1 μM MnSO4·H2O, 0.15 μM ZnSO4·7H2O, 0.16 μM CuSO4, and 0.52 μM (NH4)3Mo7O). 24 The solution is placed in a blue box containing 4H2O (pH 5.5). When treating with different nitrogen concentrations, the 1N (1.25mM NH4NO3) in the standard nutrient solution needs to be replaced with 0.6N (0.75mM NH4NO3), 0.3N (0.375mM NH4NO3), and 0.15N (0.1875mM NH4NO3). The solution is cultured for 4 weeks, with the pH adjusted every 2 days.
[0033] After 4 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 Nitrate content (obtained by Li Yuzhong's laboratory at the Chinese Academy of Agricultural Sciences, using an Isoprime 100 instrument). The results showed that the nitrate nitrogen uptake rate of the mutant osrnu9 was significantly decreased. Figure 3 e).
Claims
1. Application of rice OsRNU9 gene in improving nitrogen fertilizer use efficiency of rice, characterized in that, The nucleotide sequence of the OsRNU9 gene is shown as SEQ ID NO.
1.
2. Use according to claim 1, characterized in that, OsRNU9 responds to the external nitrogen supply level, and the expression level is induced by low concentration of nitrogen.
3. Use according to claim 1, characterized in that, Knocking out or silencing the OsRNU9 gene can reduce the nitrogen absorption rate, plant height, tillering and yield of rice.
4. The potential application value of the rice OsRNU9 gene in improving the nitrogen fertilizer use efficiency of rice in claim 1.