Application of rice osarf24 gene

Abstract

The application discloses application of a rice OsARF24 gene. The nucleotide sequence of the OsARF24 gene has an accession number of LOC4352211 in Genbank. The rice OsARF24 gene is a key element in an auxin signal pathway. Knocking out the OsARF1 can cause the expression amount of nitrogen metabolism related genes of the rice, the nitrate nitrogen absorption rate, the plant height and the yield of the rice to be reduced, and the nitrogen fertilizer utilization efficiency to be reduced in a comprehensive manner. The function of the OsARF24 gene in positively regulating the nitrogen fertilizer utilization efficiency of the rice is clarified, and a technical route for increasing the nitrogen fertilizer utilization efficiency of the rice by increasing the expression of the OsARF24 in the rice is provided.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology and discloses an application of OsARF24 in regulating nitrogen fertilizer use efficiency and yield in rice. Background Technology

[0002] Rice (Oryza sativa L.) is an important food crop. Nitrogen is a key factor affecting rice yield, and applying nitrogen fertilizer is an important means to increase grain 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), which is widely used in rice breeding, can improve the harvest index and the lodging resistance of rice, effectively solving the problem of rice lodging caused by factors such as excessive fertilization and wind and rain, thereby significantly increasing rice yield (Li et al., 2018).

[0004] While semi-dwarf rice varieties exhibit higher yields, they show insensitivity to nitrogen fertilizer, reducing nitrogen fertilizer use efficiency (Prabhu and Pingali, 2012). Auxins and nitrogen are both important signaling molecules promoting plant growth and development. Currently, our understanding of the synergistic regulation of plant growth and development by auxin and nitrogen mainly comes from Arabidopsis studies, primarily focusing on root morphology. In rice, how the auxin signaling pathway affects nitrogen fertilizer use efficiency, and how to improve nitrogen fertilizer use efficiency by modifying key components of the auxin signaling pathway, remains unclear. Summary of the Invention

[0005] The purpose of this invention is to address the aforementioned shortcomings of the prior art by providing an application of the rice OsARF24 gene.

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

[0007] The objective of this invention can be achieved through the following technical solutions:

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

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

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

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

[0012] Overexpressing the said OsARF24 gene can improve the nitrogen use efficiency and / or yield of rice.

[0013] A method for improving the nitrogen use efficiency of rice, which realizes the improvement of the nitrogen use efficiency and yield of rice by increasing the expression of OsARF24 through genetic engineering means.

[0014] Beneficial effects:

[0015] In this invention, the CRISPR / Cas9 technology is used to knock out the OsARF24 gene of rice, and the mutant material osarf24 is constructed. By measuring the nitrate nitrogen absorption rate labeled with 15 N isotope and conducting field phenotype investigation on the wild-type material and the mutant material, it is found that knocking out the OsARF24 gene will reduce the expression level of genes related to nitrogen metabolism, nitrate nitrogen absorption rate, plant height and yield of rice, clarify the function of the OsARF24 gene in positively regulating the nitrogen use efficiency of rice, and provide a technical route for improving the nitrogen use efficiency of rice by increasing the expression of OsARF24 in rice. Description of the drawings

[0016] Figure 1 Showing the mutation type of the OsARF24 gene in the mutant material osarf24. The structure of the OsARF24 gene is shown as follows. 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 Showing the expression levels of genes related to nitrogen metabolism in the wild-type ZH11 and the mutant material osarf24. (a), the transcriptional level of OsNRT1.1B; (b), the transcriptional level of OsNRT2.3a; (c), the transcriptional level of OsNPF2.4; (d), the transcriptional level of OsNIA2;

[0018] Figure 3 Showing the comparative analysis of the phenotypes of important agronomic traits of the wild-type ZH11 and the mutant material osarf24. (a), the field phenotype of the osarf24 transgenic material; (b), plant height; (c), tillering; (d), yield per plant; (e), NO3 - Absorption rate. Detailed implementation manners

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

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

[0021] To verify that OsARF24, a key regulator of the auxin signaling pathway, can regulate NO3 in rice... - To identify key sites for absorption rate, we constructed OsARF24 knockout material using the CRISPR-Cas9 system. First, we designed the OsARF24 target sequence using the CRISPR-P website. Using the transcription unit with the rice U6+U3 promoter shown in SEQ ID NO.2 as a template, we performed PCR amplification. The PCR product was recovered from the gel and ligated to a TKC vector that had been completely digested with 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 RicePlants. Rice Science; 2019, 26(2):109-117). Finally, we transformed E. coli to obtain positive transformants, which were then sequenced. The constructed OsARF24 knockout vector was transformed into the genome of Agrobacterium using the Agrobacterium-mediated transformation method. Through Agrobacterium infection of plants, the foreign gene was transferred and integrated into plant cells, resulting in the OsARF24 gene mutant osarf24. The primer sequences for constructing the OsARF24 knockout vector are detailed in Table 1.

[0022] Table 1

[0023]

[0024] Sequencing analysis revealed a 42-G base deletion in the first exon of osarf24, which ultimately led to premature termination. Figure 1 ).

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

[0026] To investigate the impact of OsARF24 gene knockout on the expression of nitrogen metabolism-related genes in rice, we used qRT-PCR experiments to detect the expression levels of nitrogen metabolism-related genes in wild-type ZH11 and the mutant material osarf24 grown hydroponically for three weeks. 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). Subsequently, RT-PCR reactions were performed according to the manufacturer's instructions (TransGen, AQ601), with the following conditions: Step 1, 94℃ pre-denaturation for 3 min; Step 2, 98℃ denaturation for 15 s; Step 3, 58℃ annealing for 15 s; Step 4, 72℃ extension for 20 s; Steps 2, 3, and 4 were performed for 45 cycles; Step 5, 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 control. The relevant RT-qPCR primer sequences are shown in Table 2.

[0027] Table 2

[0028]

[0029] The results showed that, compared with ZH11, the transcriptional 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 uptake rate and yield of mutant osarf24

[0031] In the field yield test, wild-type ZH11 and mutant material osarf24 were planted in the field (nitrogen application rate was 210 kg / ha), and various important agronomic traits were observed and statistically analyzed.

[0032] Specific statistical methods: Plant height statistics: After rice matures, the height of 15 plants are measured in the field. Number of grains per panicle statistics: After rice matures, panicles from 12 main tillers are collected in the field, and the number of grains per panicle is counted and recorded. Yield per plant statistics: After the rice is fully mature, 12 individual plants in the plot are threshed. The harvested seeds are dried at a constant temperature of 37℃ and then weighed to obtain the yield per plant. Three replicate experiments are required.

[0033] Statistical results showed that osarf24 exhibited decreased plant height, increased tillering, and weakened plant vigor, ultimately leading to a decrease in yield per plant. Figure 3 ad).

[0034] Subsequently, we studied the biotype ZH11 and the mutant material osarf24. 15 The absorption rate of nitrogen (N) was detected. - The specific method for determining the absorption rate was as follows: Seeds of ZH11 and osarf24 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 swell. The water was 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 40L nutrient solution (1.25mM NH4NO3, 0.5mM NaH2PO4·2H2O, 0.75mM K2SO4, 1mM CaCl2, 1.667mM MgSO4·7H2O, 40μM Fe-EDTA(Na), 19μM M3BO3, 9.1μM MnSO4·H2O, 0.15μM ZnSO4·7H2O, 0.16μM CuSO4, and 0.52μM (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.

[0035] After 4 weeks of cultivation, rice roots were immersed in 0.1 mM CaSO4 for 1 minute, then transferred to a nutrient solution containing 2.5 mM K15NO3 for 5 minutes, and finally transferred back to 0.1 mM CaSO4 for 1 minute. The roots were dried with filter paper or gauze, cut, dried, ground, and the 15N content was measured (performed by the laboratory of Li Yuzhong, Chinese Academy of Agricultural Sciences, using an Isoprime 100 instrument). The results showed that osarf24 contained 15N. - The absorption rate decreased significantly. Figure 3 e).

Claims

1. Rice OsARF24 The application of genes in regulating rice yield is characterized by, The aforementioned OsARF24 The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the knockout or silencing is described above. OsARF24 The gene can reduce rice yield; or overexpression of the gene described above... OsARF24 Genes can increase rice yield.

Images