Application of rice osrnu6 gene

Abstract

The application discloses application of a rice OsRNU6 gene. The nucleotide sequence of the OsRNU6 gene has an accession number of LOC4332833 in Genbank. It is found by RNA-seq that the OsRNU6 shows response to external nitrogen supply level, and it is proved by fluorescence quantitative PCR that the OsRNU6 is induced by low nitrogen. Knocking out the OsRNU6 can cause the nitrogen absorption rate, plant height, tillering and yield of rice to be reduced, and the comprehensive performance is the decrease of nitrogen fertilizer utilization efficiency. The function of the OsRNU6 gene in positively regulating the nitrogen fertilizer utilization efficiency of rice is clarified, and a technical route for improving the nitrogen fertilizer utilization efficiency of rice by increasing the expression of the OsRNU6 in rice is provided.

Description

Technical Field

[0001] The invention belongs to the technical field of plant genetic engineering and discloses an application of OsRNU6 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, serving as the staple food for more than half of the world's population. Therefore, its safe and efficient production plays a crucial role in global food security. In the early 1960s, the development of semi-dwarf wheat and rice varieties successfully resolved the dilemma between high yield and lodging, improving the harvest index and significantly increasing crop yields. These varieties inhibit gibberellin acid (GA) metabolism or signaling pathways, conferring semi-dwarf characteristics on crops, significantly improving fertilizer tolerance and harvest index. However, this also results in reduced nitrogen uptake capacity and response to nitrogen in the roots of semi-dwarf varieties, resulting in lower nitrogen use efficiency (NUE). Therefore, while semi-dwarf varieties offer high yields, their high yields rely on substantial nitrogen fertilizer inputs.

[0003] 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

[0004] The purpose of the present invention is to address the above-mentioned deficiencies in the prior art and provide an application of the rice OsRNU6 gene.

[0005] Another object of the present invention is to provide a potential method for improving nitrogen fertilizer use efficiency of rice.

[0006] The purpose of the present invention can be achieved through the following technical solutions:

[0007] The invention relates to the use of the rice OsRNU6 gene in regulating nitrogen fertilizer utilization efficiency and / or yield of rice. The accession number of the OsRNU6 gene in Genbank is LOC4332833, and its nucleotide sequence is shown in SEQ ID NO.1.

[0008] OsRNU6 responds to the external nitrogen supply level, and its expression level is induced by low nitrogen concentration.

[0009] Knocking out or silencing the OsRNU6 gene can reduce nitrogen uptake rate, plant height and yield of rice.

[0010] Knocking out or silencing the OsRNU6 gene can reduce nitrogen fertilizer use efficiency and / or yield of rice.

[0011] Overexpression of the OsRNU6 gene can improve nitrogen fertilizer utilization efficiency and / or yield of rice.

[0012] Beneficial effects:

[0013] The present invention subjected the wild-type material ZH11 to high and low nitrogen treatments, and found that the OsRNU6 gene in rice showed a response to the external nitrogen supply level by RNA-seq experiments, and proved by fluorescence quantitative PCR that OsRNU6 had a significant tendency to be induced by low nitrogen. Therefore, the mutant material OsRNU6 was constructed, and the wild-type material and the mutant material were subjected to high and low nitrogen treatments. 15 N isotope-labeled nitrogen absorption rate measurements and field phenotypic surveys found that knocking out the OsRNU6 gene reduced rice nitrogen absorption rate, plant height, tillering and yield, clarifying the function of the OsRNU6 gene in positively regulating the nitrogen fertilizer use efficiency of rice, and providing a technical route to improve the nitrogen fertilizer use efficiency of rice by increasing the expression of OsRNU6 in rice. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 Shown are the transcript abundances of OsRNU6 at different nitrogen concentrations.

[0015] Figure 2 The diagram shows the mutation type of the OsRNU6 gene in the mutant material osrnu6. The structure of the OsRNU6 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.

[0016] Figure 3 Comparative analysis of important agronomic traits of wild-type ZH11 and mutant osrnu6. (a) Field phenotype of osrnu6 transgenic material; (b) plant height; (c) tillering; (d) yield per plant; (e) nitrogen uptake rate. DETAILED DESCRIPTION

[0017] In the following examples, ZH11 is the abbreviation of Zhonghua 11.

[0018] Example 1: OsRNU6 responds to changes in external nitrogen supply levels

[0019] To preliminarily verify whether the OsRNU6 gene responds to the external nitrogen supply level, the japonica rice material ZH11 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 OsRNU6 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.

[0020] Table 1

[0021]

[0022]

[0023] The results showed that osrnu6 responded to the nitrogen supply level in the environment, and its expression level was induced by low nitrogen concentration ( Figure 1 ).

[0024] Example 2: Construction of Rice OsRNU6 Gene Mutant OsRNU6

[0025] To verify that OsRNU6 is a key site that regulates the nitrogen uptake rate of rice, we used the CRISPR-Cas9 system to construct an OsRNU6 knockout material. First, the OsRNU6 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, Escherichia coli was transformed to obtain positive transformants and sent for sequencing. The constructed OsRNU6 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 ZH11 to generate the OsRNU6 mutant osrnu6. The primer sequences for constructing the OsRNU6 knockout vector are detailed in Table 2.

[0026] Table 2

[0027]

[0028] Sequencing analysis revealed that osrnu6 inserted a base A in the fifth exon, which ultimately caused premature termination ( Figure 2 ).

[0029] Example 3: Comparative analysis of agronomic traits such as nitrogen uptake rate and yield of the mutant osrnu6

[0030] In the field yield test, the wild type ZH11 and the mutant material osrnu6 were planted in the field (nitrogen application rate was 210 kg / ha), and various important agronomic traits were observed and counted.

[0031] 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.

[0032] Statistical comparison showed that the mutant osrnu6 had reduced plant height, fewer tillers, and weaker plant growth, ultimately leading to a decrease in single-plant yield ( Figure 3 ad).

[0033] Subsequently, we investigated the expression of wild-type ZH11 and mutant osrnu6. 15 Nitrogen absorption rate was measured. The specific method for measuring nitrogen absorption rate was as follows: ZH11 and OsRNU6 seeds were disinfected with 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, 40μMFe-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.

[0034] 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 osrnu6 was significantly reduced ( Figure 3 e).

Claims

1. Knockout or Silencing OsRNU6 The application of a gene in reducing nitrogen absorption rate, plant height and yield of rice is characterized in that: The OsRNU6 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 OsRNU6 Genes that reduce nitrogen fertilizer use efficiency and / or yield in rice.

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