Application of rice OsRNU6 gene

Abstract

The invention discloses an application of a rice OsRNU6 gene. The login number of the nucleotide sequence of the OsRNU6 gene in Genbank is LOC4332833, and the login number of the nucleotide sequence of the OsRNU6 gene in Genbank is LOC4332833. RNA-seq is utilized to discover that the OsRNU6 shows response to the external nitrogen supply level, and fluorescent quantitative PCR (Polymerase Chain Reaction) proves that the OsRNU6 is induced by low nitrogen. The OsRNU6 is knocked out, so that the nitrogen absorption rate, plant height, tillering and yield of the rice are reduced, and the comprehensive performance is that the utilization efficiency of a nitrogen fertilizer is reduced. The function of the OsRNU6 gene for positively regulating the utilization efficiency of the rice nitrogen fertilizer is defined, and a technical route for improving the utilization efficiency of the rice nitrogen fertilizer 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 for regulating rice nitrogen fertilizer utilization efficiency and yield. Background Art

[0002] Rice (Oryza sativa L.) is one of the most important food crops in the world and the staple food of more than half of the world's population. Therefore, the safe and efficient production of rice plays a vital role in the world's food security. In the early 1960s, the breeding of semi-dwarf wheat and rice varieties successfully solved the contradiction between "high yield and lodging", improved the harvest index, and greatly increased the yield of crops. Varieties inhibit the metabolism or signaling pathway of gibberellin Acid (GA), giving crops semi-dwarf characteristics, greatly improving the fertilizer tolerance and harvest index of crops. However, this also caused the semi-dwarf variety's root system to have a reduced nitrogen absorption capacity and a weakened response to nitrogen, resulting in a decrease in its nitrogen use efficiency (NUE). Therefore, although the semi-dwarf variety has a high yield, its high yield depends on a large amount of nitrogen fertilizer input.

[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 goal of breeding 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 provide an application of rice OsRNU6 gene in view of the above-mentioned deficiencies in the prior art.

[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] Application of rice OsRNU6 gene in regulating rice nitrogen fertilizer utilization efficiency and / or yield, 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 the 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 through RNA-seq experiments that the OsRNU6 gene in rice showed a response to the nitrogen supply level in the outside world, and proved through fluorescent quantitative PCR that OsRNU6 had a clear 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 would reduce rice nitrogen absorption rate, plant height, tillering and yield, clarifying the function of the OsRNU6 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 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 mutation type of the OsRNU6 gene of the mutant material osrnu6 is shown. The structure of the OsRNU6 gene is shown in the figure, exons are represented by black boxes, introns are represented by lines, and 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 material osrnu6. (a) Field phenotype of osrnu6 transgenic material; (b) plant height; (c) tillering; (d) yield per plant; (e) nitrogen absorption 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 OsRNU6 gene responds to the external nitrogen supply level, we used four nitrogen concentrations (0.15N, 0.1875mM NH 4 NO 3 ; 0.3N, 0.375mM NH 4 NO 3; 0.6N, 0.75mM NH 4 NO 3 ; 1N, 1.25 mM NH 4 NO 3 ) were cultured under the conditions of 100 μg / mL and 100 μg / mL. Total RNA was extracted for reverse transcription. The transcription level of OsRNU6 was 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 reaction was then performed according to the instructions (TransGen, AQ601). The reaction conditions were as follows: first step, pre-denaturation at 94°C for 3 min; second step, denaturation at 98°C for 15 s; third step, annealing at 58°C for 15 s; fourth step, extension at 72°C for 20 s; 45 cycles were performed in the second, third and fourth steps; fifth step, melting curve analysis. Each RT-qPCR test 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] In order to verify that OsRNU6 is a key site that can regulate the nitrogen absorption rate of rice, we used the CRISPR-Cas9 system to construct the knockout material of OsRNU6. First, the OsRNU6 target sequence was designed through the CRISPR-P website, and the rice U6+U3 promoter transcription unit shown in SEQ ID NO.2 was used as a template for PCR amplification. The PCR product was recovered by gel and connected to the TKC vector that had been completely digested by 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 by Agrobacterium tumefaciens to insert the target gene into the genome of Agrobacterium tumefaciens. With the help of Agrobacterium tumefaciens infection of plants, the transfer and integration of foreign genes into plant cells was achieved, and it was introduced into japonica rice ZH11 to obtain the OsRNU6 gene mutant osrnu6. The primer sequences for constructing the OsRNU6 knockout vector are shown 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 absorption rate and yield of 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 taken in the field to measure the plant height. Number of spikelets: After the rice matures, 12 spikelets on the main tillers are taken in the field, and the number of spikelets on each spike is directly counted and recorded. Single plant yield statistics: After the rice is fully mature, 12 single plants in the plot are taken for threshing. The harvested seeds are dried at a constant temperature of 37°C and then weighed to obtain the single plant yield data. Three repeated tests are required.

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

[0033] Subsequently, we investigated the expression of wild-type ZH11 and mutant osrnu6 15 The nitrogen absorption rate was tested. The specific method for determining the nitrogen absorption rate was as follows: the seeds of ZH11 and OsRNU6 were disinfected with 20% sodium hypochlorite solution for 30 minutes. After that, they were placed in a 37°C incubator and immersed in water for 24 hours. The water was drained and transferred to a 28°C incubator for germination. After the seeds turned white, they were transferred to a hollow 96-well plate and cultured for 7 days. The seedlings with the same growth were selected and transferred to 40L of nutrient solution (1.25mM NH 4 NO 3 ,0.5mMNaH 2 PO 4 ·2H 2 O, 0.75 mM K 2 SO 4 ,1mM CaCl 2 ,1.667mM MgSO 4 7H 2 O, 40 μM Fe-EDTA (Na), 19 μM H 3 BO 3 ,9.1μM MnSO 4 ·H 2 O, 0.15 μM ZnSO 4 7H 2 O, 0.16 μM CuSO 4 , and 0.52μM (NH 4 ) 3 Mo 7 O 24 ·4H 2 O, pH 5.5). When treating with different nitrogen concentrations, the 1N (1.25 mM NH 4 NO 3 ) was replaced with 0.6N (0.75 mM NH 4 NO 3 ),0.3N(0.375mM NH 4 NO 3 ) and 0.15N (0.1875 mM NH 4 NO 3 ), cultured for 4 weeks, and adjusted the pH value every 2 days.

[0034] After 4 weeks of cultivation, the rice roots were immersed in 0.1 mM CaSO 41 min, then transferred to a 2.5 mM K 15 NO 3 nutrient solution for 5 min and finally transferred to 0.1 mM CaSO 4 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 Isoprime100 instrument). The results showed that the nitrogen absorption rate of the mutant osrnu6 was significantly reduced ( Figure 3 e).

Claims

1. Application of rice OsRNU6 gene in regulating nitrogen fertilizer utilization efficiency and / or yield of rice, characterized in that: The nucleotide sequence of the OsRNU6 gene is shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that: OsRNU6 responds to the external nitrogen supply level, and its expression level is induced by low nitrogen concentration.

3. The use according to claim 1, characterized in that: Knocking out or silencing the OsRNU6 gene can reduce the nitrogen uptake rate, plant height and yield of rice.

4. The use according to claim 1, characterized in that: Knocking out or silencing the OsRNU6 gene can reduce nitrogen fertilizer use efficiency and / or yield of rice.

5. The use according to claim 1, characterized in that: Overexpression of the OsRNU6 gene can improve nitrogen fertilizer utilization efficiency and / or yield of rice.

Images