Application of rice OsARF1 gene

Abstract

The invention discloses an application of a rice OsARF1 gene. The rice OsARF1 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 OsARF1 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 OsARF1 in rice is provided.

Description

Technical Field

[0001] The invention belongs to the technical field of plant gene engineering and discloses an application of OsARF1 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. Applying 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). Although semi-dwarf rice varieties have relatively high yields, they are insensitive to nitrogen fertilizer response, which reduces the nitrogen fertilizer utilization efficiency of rice (Prabhu and Pingali, 2012).

[0004] Auxin and nitrogen are both important signaling molecules that promote plant growth and development. Currently, our understanding of the coordinated regulation of plant growth and development by auxin and nitrogen mainly comes from research on 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 OsARF1 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] Application of rice OsARF1 gene in regulating nitrogen fertilizer utilization efficiency and / or yield of rice, wherein the nucleotide sequence of the OsARF1 gene is shown in SEQ ID NO.1.

[0009] The loss of OsARF1 will lead to reduced expression levels of nitrogen metabolism-related genes in rice, ultimately resulting in reduced nitrogen fertilizer efficiency and yield.

[0010] Knocking out or silencing the OsARF1 gene can reduce the expression level of nitrogen metabolism-related genes in rice.

[0011] Knocking out or silencing the OsARF1 gene can reduce the nitrate nitrogen absorption rate, plant height and yield of rice.

[0012] Knocking out or silencing the OsARF1 gene can reduce nitrogen fertilizer use efficiency and / or yield of rice.

[0013] Overexpression of the OsARF1 gene can improve nitrogen fertilizer utilization efficiency and / or yield of rice.

[0014] A potential method to improve nitrogen fertilizer use efficiency of rice. Increasing the expression of OsARF1 can improve nitrogen fertilizer use efficiency and yield of rice.

[0015] Beneficial effects:

[0016] The present invention uses CRISPR / Cas9 technology to knock out the rice OsARF1 gene and constructs the mutant material osarf1. 15 N isotope-labeled nitrate nitrogen absorption rate determination and field phenotypic investigation found that knocking out the OsARF1 gene would reduce the expression of rice nitrogen metabolism-related genes, nitrate nitrogen absorption rate, plant height and yield, clarifying the function of the OsARF1 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 OsARF1 in rice. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The OsARF1 gene mutation type of the mutant material osarf1 is shown in the figure. The structure of the OsARF1 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.

[0018] Figure 2 The expression levels of nitrogen metabolism-related genes in wild-type ZH11 and mutant material osarf1 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;

[0019] Figure 3 Comparative analysis of important agronomic traits of wild-type ZH11 and mutant material osarf1. (a) Field phenotype of osarf1; (b) Plant height; (c) Tillering; (d) Single plant yield; (e) NO3 - Absorption rate. DETAILED DESCRIPTION

[0020] In the following examples, ZH11 is the abbreviation of Zhonghua 11.

[0021] Example 1: Construction of rice OsARF1 gene mutant osarf1

[0022] In order to verify that OsARF1, 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 OsARF1, a key site for absorption rate. First, the OsARF1 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; LihaoWang; Qiaoyan Wang; Rongchen Wang; Yunde Zhao; Improvements of TKC TechnologyAccelerate 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 OsARF1 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 OsARF1 gene mutant osarf1. The primer sequences for constructing the OsARF1 knockout vector are shown in Table 1.

[0023] Table 1

[0024]

[0025] Sequencing analysis revealed that there was a G base deletion at 32bp in the first exon of osarf1, which ultimately caused premature termination ( Figure 1 ).

[0026] Example 2: Expression of nitrogen metabolism-related genes in the mutant osarf1

[0027] In order to detect the effect of knocking out the OsARF1 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 osarf1 cultured for three weeks. The specific detection method is: 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), 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.

[0028] Table 2

[0029]

[0030]

[0031] 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 osarf1 were significantly decreased ( Figure 2 ad).

[0032] Example 3: Comparative analysis of agronomic traits such as nitrate nitrogen absorption rate and yield of the mutant osarf1

[0033] In the field yield test, the wild type ZH11 and the mutant material osarf1 were planted in the field (nitrogen application rate was 210 kg / ha), and various important agronomic traits were observed and counted.

[0034] 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.

[0035] The statistical results showed that osarf1 had reduced plant height, increased tillering, and weakened plant growth, which ultimately led to a decrease in single-plant yield ( Figure 3 ad).

[0036] Subsequently, we investigated the expression of ZH11 and osarf1 mutants. 15 The nitrogen absorption rate was tested. - The specific method for determining the absorption rate is as follows: the seeds of ZH11 and osarf1 were disinfected with 20% sodium hypochlorite solution for 30 minutes. After that, they were placed in a 37°C incubator and soaked in water for 24 hours. The water was drained and the seeds were 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. Select seedlings with consistent growth and transfer them into a blue box containing 40 L of nutrient solution (1.25 mM NH4NO3, 0.5 mM NaH2PO4·2H2O, 0.75 mM K2SO4, 1 mM CaCl2, 1.667 mM MgSO4·7H2O, 40 μM Fe-EDTA(Na), 19 μM H3BO3, 9.1 μM MnSO4·H2O, 0.15 μM ZnSO4·7H2O, 0.16 μM CuSO4, and 0.52 μM (NH4)3Mo7O24·4H2O, 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), cultured for 4 weeks, and the pH value was adjusted every 2 days.

[0037] 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 NO3 in osarf1 - The absorption rate decreased significantly ( Figure 3 e).

Claims

1. Application of rice OsARF1 gene in regulating nitrogen fertilizer utilization efficiency and / or yield of rice, characterized in that: The nucleotide sequence of the OsARF1 gene is shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that: Knocking out or silencing the OsARF1 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 OsARF1 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 OsARF1 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 OsARF1 gene can improve nitrogen fertilizer utilization efficiency and / or yield of rice.

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