Application of rice OsARF24 gene

Abstract

The invention discloses an application of a rice OsARF24 gene. The login number of the nucleotide sequence of the OsARF24 gene in Genbank is LOC4352211, and the login number of the nucleotide sequence of the OsARF24 gene in Genbank is LOC4352211. The rice OsARF24 gene is a key element in an auxin signal path. Knockout of the OsARF1 can cause reduction of the expression quantity, the nitrate nitrogen absorption rate, the plant height and the yield of the nitrogen metabolism related genes of the rice, and the comprehensive performance is that the utilization efficiency of the nitrogen fertilizer is reduced. The function of the OsARF24 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 OsARF24 in rice is provided.

Description

Technical Field

[0001] The invention belongs to the technical field of plant gene engineering and discloses an application of OsARF24 for regulating rice nitrogen fertilizer utilization efficiency and yield. Background Art

[0002] Rice (Oryza sativa L.) is an important food crop. Nitrogen is a key factor affecting rice yield, and the application of nitrogen fertilizer is an important means to increase food yield. However, excessive application of nitrogen fertilizer has negative impacts such as increased production costs for farmers and environmental pollution.

[0003] The semi-dwarf gene (sd1) is widely used in rice breeding. It can not only improve the harvest index, but also improve the lodging resistance of rice. It effectively solves the problem of rice plant lodging caused by excessive fertilization, wind and rain, and other factors, thereby greatly increasing rice yield (Li et al., 2018).

[0004] Although semi-dwarf rice varieties have relatively high yields, they are insensitive to nitrogen fertilizer, which reduces the nitrogen fertilizer use efficiency of rice (Prabhu and Pingali, 2012). Auxin and nitrogen are both important signaling molecules that promote plant growth and development. At present, the understanding of the coordinated regulation of plant growth and development by auxin and nitrogen mainly comes from the research of Arabidopsis, most of which focuses on root morphology and structure. In rice, it is still unclear how the auxin signaling pathway affects the nitrogen fertilizer use efficiency of rice and how to improve the nitrogen fertilizer use efficiency of rice by improving the key components of the auxin signaling pathway. Summary of the invention

[0005] The purpose of the present invention is to provide an application of rice OsARF24 gene in view of the above-mentioned deficiencies in the prior art.

[0006] Another object of the present invention is to provide a potential method for improving nitrogen fertilizer use efficiency of rice.

[0007] The purpose of the present invention can be achieved through the following technical solutions:

[0008] The invention discloses an application of rice OsARF24 gene in regulating nitrogen fertilizer utilization efficiency and / or yield of rice. The accession number of the OsARF24 gene in Genbank is LOC4352211, and the nucleotide sequence thereof is shown in SEQ ID NO.1.

[0009] Knocking out or silencing the OsARF24 gene can reduce the expression level of nitrogen metabolism-related genes in rice.

[0010] Knocking out or silencing the OsARF24 gene can reduce the nitrate nitrogen absorption rate, plant height and yield of rice.

[0011] Knocking out or silencing the OsARF24 gene can reduce nitrogen fertilizer use efficiency and / or yield of rice.

[0012] Overexpression of the OsARF24 gene can improve nitrogen fertilizer utilization efficiency and / or yield of rice.

[0013] A method for improving nitrogen fertilizer utilization efficiency of rice, which improves the expression of OsARF24 by genetic engineering means, thereby improving the nitrogen fertilizer utilization efficiency and yield of rice.

[0014] Beneficial effects:

[0015] The present invention uses CRISPR / Cas9 technology to knock out the rice OsARF24 gene and constructs the mutant material osarf24. 15 N isotope-labeled nitrate nitrogen absorption rate determination and field phenotypic investigation found that knocking out the OsARF24 gene would reduce the expression of rice nitrogen metabolism-related genes, nitrate nitrogen absorption rate, plant height and yield, clarifying the function of the OsARF24 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 OsARF24 in rice. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The figure shows the mutation type of the OsARF24 gene of the mutant material osarf24. The structure of the OsARF24 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.

[0017] Figure 2 The expression levels of nitrogen metabolism-related genes in wild-type ZH11 and mutant material osarf24 are shown. (a) Transcription level of OsNRT1.1B; (b) Transcription level of OsNRT2.3a; (c) Transcription level of OsNPF2.4; (d) Transcription level of OsNIA2;

[0018] Figure 3 Comparative analysis of important agronomic traits of wild-type ZH11 and mutant material osarf24. (a) Field phenotype of osarf24 transgenic material; (b) Plant height; (c) Tillering; (d) Single plant yield; (e) NO3 - Absorption rate. DETAILED DESCRIPTION

[0019] In the following examples, ZH11 is the abbreviation of Zhonghua 11.

[0020] Example 1: Construction of rice OsARF24 gene mutant osarf24

[0021] In order to verify that OsARF24, a key regulator of the auxin signaling pathway, is able to regulate rice NO3 - We used the CRISPR-Cas9 system to construct knockout materials for OsARF24, a key site for absorption rate. First, the OsARF24 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 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, Escherichia coli was transformed to obtain positive transformants and sent for sequencing. The constructed OsARF24 knockout vector was transformed by Agrobacterium to insert the target gene into the genome of Agrobacterium. With the help of Agrobacterium 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 OsARF24 gene mutant osarf24. The primer sequences for constructing the OsARF24 knockout vector are shown in Table 1.

[0022] Table 1

[0023]

[0024] Sequencing analysis revealed that osarf24 had a 42-G base deletion in the first exon, which ultimately caused premature termination ( Figure 1 ).

[0025] Example 2: Expression of nitrogen metabolism-related genes in the mutant osarf24

[0026] In order to detect the effect of knocking out the OsARF24 gene on the expression of genes related to nitrogen metabolism in rice, we used qRT-PCR experiments to detect the expression of nitrogen metabolism-related genes using wild-type ZH11 and mutant material osarf24 that had been hydroponically cultivated for three weeks. The specific detection method was: 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 reactions were then performed according to the instructions (TransGen, AQ601), and the reaction conditions were: 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, third and fourth steps were performed for 45 cycles; 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 2.

[0027] Table 2

[0028]

[0029] The results showed that compared with ZH11, the transcription levels of nitrogen metabolism-related genes OsNRT1.1B, OsNRT2.3a, OsNPF2.4 and OsNIA2 in the mutant material osarf24 were significantly decreased ( Figure 2 ad).

[0030] Example 3: Comparative analysis of agronomic traits such as nitrate nitrogen absorption rate and yield of mutant osarf24

[0031] In the field yield test, the wild type ZH11 and the mutant material osarf24 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 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.

[0033] The statistical results showed that osarf24 had reduced plant height, increased tillering, and weakened plant growth, which ultimately led to a decrease in single-plant yield ( Figure 3 ad).

[0034] Subsequently, we investigated the expression of ZH11 and osarf24 mutants. 15 The nitrogen absorption rate was tested. - The specific method for determining the absorption rate is as follows: the seeds of ZH11 and osarf24 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. Drain the water and transfer to a 28°C incubator for germination. After turning white, they were transferred to a hollow 96-well plate and cultured for 7 days. Select seedlings with consistent growth and transfer them 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). When treated 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 culture was continued for 4 weeks and the pH value was adjusted every 2 days.

[0035] After 4 weeks of cultivation, the rice roots were immersed in 0.1mM CaSO4 for 1 minute, then transferred to a nutrient solution containing 2.5mM K15NO3 for 5 minutes, and finally transferred to 0.1mM CaSO4 for 1 minute. The roots were dried with filter paper or gauze, cut, dried, ground, and the 15N content was measured (completed by Li Yuzhong's laboratory at the Chinese Academy of Agricultural Sciences, using an Isoprime 100 instrument). The results showed that the NO3 - The absorption rate decreased significantly ( Figure 3 e).

Claims

1. Application of rice OsARF24 gene in regulating nitrogen fertilizer utilization efficiency and / or yield of rice, characterized in that: The nucleotide sequence of the OsARF24 gene is shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that: Knocking out or silencing the OsARF24 gene can reduce the expression level of nitrogen metabolism-related genes in rice.

3. The use according to claim 1, characterized in that: Knocking out or silencing the OsARF24 gene can reduce the nitrate nitrogen absorption rate, plant height and yield of rice.

4. The use according to claim 1, characterized in that: Knocking out or silencing the OsARF24 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 OsARF24 gene can improve nitrogen fertilizer utilization efficiency and / or yield of rice.

6. A method for improving nitrogen fertilizer utilization efficiency of rice, characterized in that: By increasing the expression of OsARF24 through genetic engineering, the nitrogen fertilizer utilization efficiency and yield of rice can be improved.

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