Application of rice OsNMG10 gene

Abstract

The invention discloses an application of a rice OsNMG10 gene. The registration number of the nucleotide sequence of the OsNMG10 gene in Genbank is LOC4349348, and the registration number of the nucleotide sequence of the OsNMG10 gene in Genbank is LOC4349348. The OsNMG10 is a key gene influencing the development of a rice root system. The OsNMG10 is knocked out, so that the nitrate nitrogen absorption rate, the plant height and the yield of the rice are reduced, and the comprehensive performance is that the nitrogen fertilizer utilization efficiency is reduced. The function of the OsNMG10 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 OsNMG10 in rice is provided.

Description

Technical Field

[0001] The invention belongs to the technical field of plant genetic engineering and discloses an application of OsNMG10 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, but excessive application of nitrogen fertilizer has negative effects such as increased production costs 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 resistance of rice to lodging. It effectively solves the problem of rice plant lodging caused by excessive fertilization, wind and rain, and other factors, thereby greatly increasing rice yield. Although semi-dwarf rice varieties have relatively high yields, they are insensitive to nitrogen fertilizer, which reduces the nitrogen fertilizer utilization efficiency of rice.

[0004] The cell wall is a characteristic structure of plant cells. Traditional knowledge believes that the cell wall is an inactive cell structure. Recent studies have shown that the cell wall may be an active and dynamically changing "organelle" that can sense environmental and developmental signals, or produce some signal molecules, playing an important role in cell growth, differentiation, and immune response. Cell wall relaxants have important biological functions in cell growth, affect the root structure and plant height of rice, and play a key role in the formation of root hairs. The absorption of nitrogen by rice mainly acts on the root system. In rice, how cell wall relaxants affect the nitrogen fertilizer utilization efficiency of rice is still unclear. Summary of the invention

[0005] The purpose of the present invention is to provide an application of rice OsNMG10 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 OsNMG10 gene in regulating nitrogen fertilizer utilization efficiency and / or yield of rice. The accession number of the nucleotide sequence of the OsNMG10 gene in Genbank is LOC4349348, and the nucleotide sequence is shown in SEQ ID NO.1.

[0009] The expression of OsNMG10 gene was induced by high nitrogen.

[0010] Knocking out or silencing the OsNMG10 gene can reduce the nitrate nitrogen absorption rate, plant height and yield of rice.

[0011] Knocking out or silencing the OsNMG10 gene can reduce nitrogen fertilizer use efficiency and / or yield of rice.

[0012] Overexpression of the OsNMG10 gene can improve nitrogen fertilizer utilization efficiency and / or yield of rice.

[0013] Beneficial effects:

[0014] The present invention subjected the wild-type material WYJ7-DEP1 to high and low nitrogen treatments, and found through RNA-seq experiments that the OsNMG10 gene in rice showed a response to the nitrogen supply level in the outside world, and proved through fluorescent quantitative PCR that OsNMG10 had a significant tendency to be induced by high nitrogen. Therefore, the mutant material osnmg10 was constructed, and the wild-type material and the mutant material were subjected to high nitrogen treatment. 15 N isotope-labeled nitrogen absorption rate measurements and field phenotypic surveys found that knocking out the OsNMG10 gene would reduce rice nitrogen absorption rate, plant height, and yield, clarifying the function of the OsNMG10 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 OsNMG10 in rice. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The transcript levels of OsNMG10 in WYJ7-DEP1 under culture conditions of different nitrogen concentrations are shown;

[0016] Figure 2 The figure shows the mutation type of the OsNMG10 gene of the mutant material osnmg10. The structure of the OsNMG10 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 3 Comparative analysis of important agronomic traits of wild-type WYJ7-DEP1 and mutant material osnmg10. (A) Field phenotype of osnmg10 transgenic material; (B) Plant height; (C) Tillering; (D) Single plant yield; (E) NO3 - Absorption rate. DETAILED DESCRIPTION

[0018] In the following examples, WYJ7 is the abbreviation of Wuyunjing No. 7.

[0019] Example 1: OsNMG10 responds to changes in external nitrogen supply levels

[0020] In order to find genes in rice that are induced by external nitrogen levels, we treated the wild-type material WYJ7-DEP1 with high and low nitrogen (1N, 1.25mM NH4NO3; 0.6N, 0.75mM NH4NO3; 0.3N, 0.375mMNH4NO3; 0.15N, 0.1875mM NH4NO3), and used RNA-seq experiments to find that the OsNMG10 gene in rice showed a response to the external nitrogen supply level, and qRT-PCR detection showed that OsNMG10 had a clear trend of being induced by high nitrogen.

[0021] The specific detection method is: total RNA is extracted from different plant tissues using TRIzol reagent, and full-length cDNA is 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°C pre-denaturation for 3min; second step, 98°C denaturation for 15s; third step, 58°C annealing for 15s; fourth step, 72°C extension for 20s; second, third and fourth steps for 45 cycles; fifth step, melting curve analysis. Each RT-qPCR assay included at least three biological replicates. Rice ACTIN1 gene (OsActin1, LOC_Os03g50885) was used as an internal reference. The relevant RT-qPCR primer sequences are shown in Table 1.

[0022] Table 1

[0023]

[0024] The results showed that under low nitrogen concentration culture conditions, the transcript level of gene OsNMG10 in the wild-type WYJ7-DEP1 material was significantly decreased ( Figure 1 ).

[0025] Example 2: Construction of rice OsNMG10 gene mutant osnmg10

[0026] In order to verify that OsNMG10 is able to regulate rice NO3 -We used the CRISPR-Cas9 system to construct knockout materials of OsNMG10 for the key sites of absorption rate. First, the OsNMG10 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 Isolationof 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 OsNMG10 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 WYJ7-DEP1 to obtain the OsNMG10 gene mutant osnmg10. The primer sequences for constructing the OsNMG10 knockout vector are shown in Table 2.

[0027] Table 2

[0028]

[0029] Sequencing analysis revealed that osnmg10 had a deletion of one A base in the first exon, which ultimately caused premature termination ( Figure 2 ).

[0030] Example 3: Comparative analysis of agronomic traits such as nitrate nitrogen absorption rate and yield of mutant osnmg10

[0031] In the field yield test, the wild type WYJ7-DEP1 and the mutant material osnmg10 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 osnmg10 had reduced plant height, fewer tillers, and weaker plant growth, which ultimately led to a decrease in single-plant yield ( Figure 3 AD).

[0034] Subsequently, we investigated the wild-type WYJ7-DEP1 and OsNMG10 mutant materials. 15 The nitrogen absorption rate was tested. - The specific method for determining the absorption rate is as follows: the seeds of WYJ7-DEP1 and osnmg10 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 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 to 40L nutrient solution (1.25mM NH4NO3, 0.5mM NaH2PO4·2H2O, 0.75mM K2SO4, 1mM CaCl2, 1.667mM MgSO4·7H2O, 40μM Fe-EDTA(Na), 19μM H3BO3, 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 pH value was adjusted every 2 days for 4 weeks.

[0035] After 4 weeks of cultivation, the rice roots were immersed in 0.1 mM CaSO4 for 1 min 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 Isoprime100). The results showed that osnmg10 of NO3 - The absorption rate decreased significantly ( Figure 3 E).

Claims

1. Application of rice OsNMG10 gene in regulating nitrogen fertilizer utilization efficiency and / or yield of rice, characterized in that: The nucleotide sequence of the OsNMG10 gene is shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that: The expression of OsNMG10 gene was induced by high nitrogen.

3. The use according to claim 1, characterized in that: Knocking out or silencing the OsNMG10 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 OsNMG10 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 OsNMG10 gene can improve nitrogen fertilizer utilization efficiency and / or yield of rice.

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