Application of rice OsNMG12 gene

Abstract

The invention discloses an application of a rice OsNMG12 gene. The login number of the nucleotide sequence of the OsNMG12 gene in Genbank is LOC4352424, and the number of the nucleotide sequence of the OsNMG12 gene in Genbank is LOC4352424. RNA-seq is utilized to find that the OsNMG12 shows response to the external nitrogen supply level, and fluorescent quantitative PCR (Polymerase Chain Reaction) proves that the OsNMG12 is induced by low nitrogen. Knockout of the OsNMG12 can cause reduction of the nitrogen absorption rate, plant height, tillering and yield of the rice, and the comprehensive performance is that the utilization efficiency of the nitrogen fertilizer is reduced. The function of the OsNMG12 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 OsNMG12 in rice is provided.

Description

Technical Field

[0001] The invention belongs to the technical field of plant genetic engineering and discloses an application of OsNMG12 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. At present, the global population is growing rapidly. According to the forecast of the Population Division of the United Nations Department of Economic and Social Affairs, the world population will grow to 10.9 billion in 2100. The "State of Food Security and Nutrition in the World 2020" pointed out that the number of hungry people in the world continues to rise, and more than 690 million people were hungry in 2019. The contradiction between population growth and food supply remains prominent. To meet the needs of human development in the next 30 years, the world's food production needs to increase by at least 70%.

[0003] 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, greatly increased the yield of crops, and greatly alleviated the food crisis caused by rapid population growth. This was called the first "Green Revolution". The "Green Revolution" varieties inhibited the metabolism or signaling pathway of gibberellin Acid (GA), endowed crops with semi-dwarf characteristics, and greatly improved the fertilizer tolerance and harvest index of crops.

[0004] However, this also results in a decrease in the nitrogen absorption capacity of the roots of semi-dwarf varieties and a weakened response to nitrogen, resulting in a decrease in their nitrogen use efficiency (NUE). Therefore, although the yield of semi-dwarf varieties is higher, their high yield depends on a large amount of nitrogen fertilizer input.

[0005] 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

[0006] The purpose of the present invention is to provide an application of rice OsNMG12 gene in view of the above-mentioned deficiencies in the prior art.

[0007] Another object of the present invention is to provide a potential method for improving nitrogen fertilizer use efficiency of rice.

[0008] The purpose of the present invention can be achieved through the following technical solutions:

[0009] Application of rice OsNMG12 gene in regulating nitrogen fertilizer utilization efficiency of rice. The accession number of the OsNMG12 gene in Genbank is LOC4352424, and the nucleotide sequence is shown in SEQ ID NO.1.

[0010] OsNMG12 responds to the external nitrogen supply level, and its expression level is induced by low nitrogen concentration.

[0011] Knocking out or silencing the OsNMG12 gene can reduce the nitrogen uptake rate, plant height and yield of rice.

[0012] Knocking out or silencing the OsNMG12 gene can reduce the nitrogen use efficiency of rice.

[0013] Overexpression of the OsNMG12 gene can improve the nitrogen fertilizer utilization efficiency of rice.

[0014] Beneficial effects:

[0015] The present invention subjected the wild-type material ZH11 to high and low nitrogen treatments, analyzed and compared the RNA-seq data of the cultured plants under different nitrogen supply levels, and found that the expression of OsNMG12 in rice responded to the external nitrogen supply level. Further fluorescence quantitative PCR proved that OsNMG12 had a significant tendency to be induced by low nitrogen. Therefore, the mutant material osnmg12 was constructed. 15 N isotope-labeled nitrogen absorption rate measurements and field phenotypic surveys found that knocking out the OsNMG12 gene would reduce rice nitrogen absorption rate, plant height, tillering and yield, clarifying the function of the OsNMG12 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 OsNMG12 in rice. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Shown are the transcript abundances of OsNMG12 under different nitrogen concentrations.

[0017] Figure 2 The mutation type of the OsNMG12 gene of the mutant material osnmg12 is shown in the figure. The structure of the OsNMG12 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 3 Comparative analysis of important agronomic traits of wild-type ZH11 and mutant material osnmg12. (a) Field phenotype of osnmg12 transgenic material; (b) plant height; (c) tillering; (d) yield per plant; (e) nitrogen absorption rate. DETAILED DESCRIPTION

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

[0020] Example 1: OsNMG12 responds to changes in external nitrogen supply levels

[0021] In order to preliminarily verify whether the OsNMG12 gene responds to the external nitrogen supply level, the japonica rice material ZH11 cultured under four nitrogen concentrations (0.15N, 0.1875mM NH4NO3; 0.3N, 0.375mM NH4NO3; 0.6N, 0.75mM NH4NO3; 1N, 1.25mM NH4NO3) was used to extract its total RNA for reverse transcription, and then the transcription level of OsNMG12 was detected by fluorescent quantitative PCR, and its expression level was analyzed 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), and the reaction conditions were: first step, 94 ° C pre-denaturation for 3 min; second step, 98 ° C denaturation for 15 s; third step, 58 ° C annealing for 15 s; fourth step, 72 ° C extension for 20 s; second step, third step and fourth step for 45 cycles; fifth step, melting curve analysis. Each RT-qPCR test 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 OsNMG12 responded to the nitrogen supply level in the environment, and its expression level was induced by low nitrogen concentration ( Figure 1 ).

[0025] Example 2: Construction of rice OsNMG12 gene mutant osnmg12

[0026] In order to verify that OsNMG12 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 OsNMG12. First, the OsNMG12 target sequence was designed through the CRISPR-P website, and the rice U6+U3 promoter transcription unit shown in SEQ ID.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 cut 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 OsNMG12 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 OsNMG12 gene mutant osnmg12. The primer sequences for constructing the OsNMG12 knockout vector are shown in Table 2.

[0027] Table 2

[0028]

[0029] Sequencing analysis revealed that osnmg12 had a G base deletion in the second 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 osnmg12

[0031] In the field yield test, the wild type ZH11 and the mutant material osnmg12 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 osnmg12 had lower 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 expression of wild-type ZH11 and mutant osnmg12 15 The nitrogen absorption rate was tested. The specific method for determining the nitrogen absorption rate was as follows: the seeds of ZH11 and osnmg12 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 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 0.1 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 Isoprime 100). The results showed that the nitrogen absorption rate of osnmg12 decreased significantly ( Figure 3 e).

Claims

1. Application of rice OsNMG12 gene in regulating nitrogen fertilizer utilization efficiency of rice, characterized in that: The nucleotide sequence of the OsNMG12 gene is shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that: OOsNMG12 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 OsNMG12 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 OsNMG12 gene can reduce the nitrogen use efficiency of rice.

5. The use according to claim 1, characterized in that: Overexpression of the OsNMG12 gene can improve the nitrogen fertilizer utilization efficiency of rice.

Images