Application of Rice OsRNU8 Gene

Abstract

The present invention discloses the application of the rice OsRNU8 gene. The nucleotide sequence of the OsRNU8 gene is registered in Genbank as LOC4333925. RNA-seq revealed that OsRNU8 responds to external nitrogen supply levels, and fluorescence quantitative PCR demonstrated that OsRNU8 is induced by high nitrogen. Knockout of OsRNU8 results in reduced nitrogen uptake rate, plant height, and yield in rice, which is manifested as a decrease in nitrogen fertilizer use efficiency. The OsRNU8 gene's function in positively regulating rice nitrogen fertilizer use efficiency has been clarified, providing a technical approach for improving rice nitrogen fertilizer use efficiency by increasing the expression of OsRNU8 in rice.

Description

Technical Field

[0001] The invention belongs to the technical field of plant genetic engineering and discloses an application of OsRNU8 in regulating nitrogen fertilizer utilization efficiency and yield of rice. Background Art

[0002] Rice (Oryza sativa L.) is one of the world's most important food crops and the staple food for more than half of the world's population. Therefore, safe and efficient rice production plays a crucial role in global food security. However, the contradiction between population growth and food supply remains prominent.

[0003] In the early 1960s, the development of semi-dwarf wheat and rice varieties successfully resolved the conflict between high yield and lodging, improving the harvest index and significantly increasing crop yields per unit area, significantly alleviating the food crisis brought on by rapid population growth. These "Green Revolution" varieties inhibited gibberellin acid (GA) metabolism or signaling pathways, imparting semi-dwarf characteristics to crops and significantly improving their fertilizer tolerance and harvest index.

[0004] However, this also results in a decrease in the nitrogen uptake capacity of the roots of semi-dwarf varieties, weakening their nitrogen response, resulting in a lower nitrogen use efficiency (NUE). Therefore, although semi-dwarf varieties have high yields, their high yields rely on large amounts of nitrogen fertilizer. Since the 1970s, my country's grain production has shown a sustained growth trend, but this is mainly due to the continuous increase in nitrogen fertilizer application.

[0005] Although a number of genes that can improve rice NUE have been identified, there is still a long way to go to achieve the cultivation of truly "green and high-yield" rice varieties. It is still necessary to continuously explore new genetic resources and analyze and identify the regulatory networks in plant development and nutrient metabolism. Summary of the Invention

[0006] The purpose of the present invention is to address the above-mentioned deficiencies in the prior art and provide an application of the rice OsRNU8 gene.

[0007] Another object of the present invention is to provide a potential method for improving nitrogen fertilizer use efficiency of rice.

[0008] The purpose of the present invention can be achieved through the following technical solutions:

[0009] The invention relates to the application of the rice OsRNU8 gene in regulating nitrogen fertilizer utilization efficiency of rice. The accession number of the OsRNU8 gene in Genbank is LOC4333925, and its nucleotide sequence is shown in SEQ ID NO.1.

[0010] OsRNU8 responds to the external nitrogen supply level, and its expression level is induced by high nitrogen concentration.

[0011] Knocking out or silencing the OsRNU8 gene can reduce nitrogen uptake rate, plant height and yield of rice.

[0012] Knocking out or silencing the OsRNU8 gene can reduce nitrogen use efficiency and / or yield.

[0013] The use according to claim 1, characterized in that overexpression of the OsRNU8 gene can improve nitrogen fertilizer utilization efficiency and / or yield.

[0014] Beneficial effects:

[0015] The present invention subjected the wild-type material WYJ7 to high and low nitrogen treatments, and found that the OsRNU8 gene in rice showed a response to the external nitrogen supply level by RNA-seq experiments, and proved by fluorescence quantitative PCR that OsRNU8 had a significant tendency to be induced by high nitrogen. Therefore, the mutant material OsRNU8 was constructed, and the wild-type material and the mutant material were subjected to high nitrogen treatment. 15 N isotope-labeled nitrogen absorption rate measurements and field phenotypic surveys found that knocking out the OsRNU8 gene would reduce the nitrogen absorption rate, plant height and yield of rice, clarifying the function of the OsRNU8 gene in positively regulating the nitrogen fertilizer utilization efficiency of rice, and providing a technical route to improve the nitrogen fertilizer utilization efficiency of rice by increasing the expression of OsRNU8 in rice. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Shown are the transcript abundances of OsRNU8 at different nitrogen concentrations.

[0017] Figure 2 The diagram shows the mutation type of the OsRNU8 gene in the mutant material osrnu8. The structure of the OsRNU8 gene is shown in the figure, with exons represented by black boxes and introns by lines. The nucleic acid sequence below the gene structure is the target sequence.

[0018] Figure 3 Comparative analysis of important agronomic traits of wild-type WYJ7 and mutant osrnu8. (a) Field phenotype of osrnu8 transgenic material; (b) plant height; (c) tillering; (g) yield per plant; (h) nitrogen uptake rate. DETAILED DESCRIPTION

[0019] In the following examples, WYJ7 is the abbreviation of Wuyunjing 7.

[0020] Example 1: OsRNU8 responds to changes in external nitrogen supply levels

[0021] To preliminarily verify whether the OsRNU8 gene responds to the external nitrogen supply level, the japonica rice material WYJ7 was cultured under four nitrogen concentrations (0.15N, 0.1875mM NH4NO3; 0.3N, 0.375mM NH4NO3; 0.6N, 0.75mM NH4NO3; 1N, 1.25mM NH4NO3). The total RNA was extracted and reverse transcribed. The transcription level of OsRNU8 was then detected by fluorescence quantitative PCR, and its expression level was analyzed to see whether it changed with the change of external N concentration. The specific detection method was as follows: total RNA was extracted from different plant tissues using TRIzol reagent, and full-length cDNA was reverse transcribed using a cDNA synthesis kit (TransGen, AT341). RT-PCR was then performed according to the manufacturer's instructions (TransGen, AQ601). The reaction conditions were as follows: first, pre-denaturation at 94°C for 3 minutes; second, denaturation at 98°C for 15 seconds; third, annealing at 58°C for 15 seconds; fourth, extension at 72°C for 20 seconds; 45 cycles of steps 2, 3, and 4; and fifth, melting curve analysis. Each RT-qPCR assay included at least three biological replicates. The rice ACTIN1 gene (OsActin1, LOC_Os03g50885) was used as an internal reference. The relevant RT-qPCR primer sequences are shown in Table 1.

[0022] Table 1

[0023]

[0024] The results showed that OsRNU8 responded to the nitrogen supply level in the environment, and its expression level was induced by high nitrogen concentration ( Figure 1 ).

[0025] Example 2: Construction of rice OsRNU8 gene mutant osrnu8

[0026] To verify that OsRNU8 is a key site that regulates the nitrogen uptake rate of rice, we used the CRISPR-Cas9 system to construct knockout materials for OsRNU8. First, the OsRNU8 target sequence was designed using the CRISPR-P website. PCR amplification was performed using the rice U6+U3 promoter transcription unit shown in SEQ ID NO.2 as a template. The PCR product was recovered from the gel and ligated to the TKC vector that had been completely digested with SpeI (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, positive transformants were transformed into Escherichia coli and sent for sequencing. The constructed OsRNU8 knockout vector was transformed with Agrobacterium tumefaciens to insert the target gene into the genome. With the help of Agrobacterium infection, the exogenous gene was transferred and integrated into plant cells. The gene was then introduced into japonica rice WYJ7 to generate the OsRNU8 mutant osrnu8. The primer sequences for constructing the osrnu8 knockout vector are detailed in Table 2.

[0027] Table 2

[0028]

[0029] Sequencing analysis revealed that OsRNU8 inserted a base T in the second exon, which ultimately caused premature termination ( Figure 2 ).

[0030] Example 3: Comparative analysis of agronomic traits such as nitrogen uptake rate and yield of the mutant osrnu8

[0031] In the field yield test, the wild type WYJ7 and the mutant material osrnu8 were planted in the field (nitrogen application rate was 210 kg / ha), and various important agronomic traits were observed and counted.

[0032] Specific statistical methods: Plant height statistics: After the rice matures, 15 plants are collected in the field to measure their plant height. Number of grains per panicle: After the rice matures, 12 panicles on the main tillers are collected in the field, and the number of grains on each panicle is directly counted and recorded. Single plant yield statistics: After the rice is fully mature, 12 single plants in the plot are threshed. The harvested seeds are dried at a constant temperature of 37°C and then weighed to obtain the single plant yield data. The test needs to be repeated three times.

[0033] Statistical comparison showed that the mutant osrnu8 had reduced plant height, increased tillering, and weakened plant growth, ultimately leading to a decrease in single-plant yield ( Figure 3 ad).

[0034] Subsequently, we investigated the expression of wild-type WYJ7 and mutant osrnu8. 15 The nitrogen absorption rate was tested. The specific method for measuring nitrogen absorption rate was as follows: WYJ7 and OsRNU8 seeds were disinfected with a 20% sodium hypochlorite solution for 30 minutes. Then, they were placed in a 37°C incubator and allowed to swell in water for 24 hours. The seeds were drained and transferred to a 28°C incubator for germination. After the seeds appeared white, they were transferred to a 96-well plate with openwork holes and cultured for 7 days. The seedlings with the same growth were selected and transferred to 40L nutrient solution (1.25mM NH4NO3, 0.5mM NaH2PO4·2H2O, 0.75mM K2SO4, 1mM CaCl2, 1.667mM MgSO4·7H2O, 40μM Fe-EDTA(Na), 19μMH3BO3, 9.1μM MnSO4·H2O, 0.15μM ZnSO4·7H2O, 0.16μM CuSO4, and 0.52μM (NH4)3Mo7O). 24 4H2O, pH 5.5). For different nitrogen concentration treatments, the 1N (1.25mM NH4NO3) in the standard nutrient solution was replaced with 0.6N (0.75mM NH4NO3), 0.3N (0.375mM NH4NO3), and 0.15N (0.1875mM NH4NO3). Cultures were maintained for four weeks, with the pH adjusted every two days.

[0035] After 4 weeks of culture, the rice roots were immersed in 0.1 mM CaSO4 for 1 minute and then transferred to a medium containing 2.5 mM K 15 NO3 nutrient solution for 5 minutes, and finally transferred to 0.1mM CaSO4 for 1 minute. Use filter paper or gauze to absorb the moisture of the roots, cut the roots, dry them, grind them, and measure 15 N content (completed by Li Yuzhong's laboratory at the Chinese Academy of Agricultural Sciences, using the Isoprime 100 instrument). The results showed that the nitrogen absorption rate of the mutant osrnu8 was significantly reduced ( Figure 3 e).

Claims

1. Knockout or Silencing OsRNU8 The application of a gene in reducing nitrogen absorption rate, plant height and yield of rice is characterized in that: The OsRNU8 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that Knockout or silencing of OsRNU8 Genes that reduce nitrogen use efficiency and / or yield.

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