Application of OsNER1 gene in regulation and control of nitrogen utilization efficiency and yield of rice

Abstract

The invention provides application of an OsNER1 gene in regulation and control of nitrogen utilization efficiency and yield of rice, a nucleotide sequence of the OsNER1 gene is shown as SEQ ID NO.1, and the gene negatively regulates and controls nitrogen transport capacity of the rice under a low-nitrogen culture condition. Under the low-nitrogen culture condition, the overground part height and biomass of the Oser1 mutant plant are remarkably increased, and the inhibition of low-nitrogen stress on plant growth is effectively overcome. The 15N content ratio of the overground part to the underground part of the Oser1 mutant plant is obviously higher than that of a wild plant, which indicates that the nitrogen absorption or transport capability of the plant under a low-nitrogen condition is enhanced by the deletion of the OsNER1 gene.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of plant breeding and biology, and particularly relates to OsNER1 Application of a gene in regulating nitrogen utilization efficiency and yield of rice. BACKGROUND

[0003] Food security is an important cornerstone of national strategy. As the main global food crop, the sustainable and high yield of rice is of great significance to guarantee food supply. Nitrogen is an indispensable mineral element in the growth and development of rice. As an important component of life substances such as protein, chlorophyll and nucleic acid, it directly affects the physiological metabolism and yield formation of plants. However, in current rice cultivation, the phenomenon of blindly increasing nitrogen fertilizer for the purpose of high yield is common, which not only increases the production cost, but also causes about 60%-70% of nitrogen fertilizer to enter the environment without being absorbed by crops, leading to ecological problems such as water eutrophication and soil acidification. Therefore, improving nitrogen utilization efficiency and realizing "reducing nitrogen without reducing yield" have become an urgent need for the sustainable development of global rice production.

[0004] In breeding practice, breeding nitrogen-efficient varieties is considered to be a fundamental way to improve nitrogen utilization rate. However, due to the involvement of multiple genes in the regulation network of nitrogen absorption, transport and assimilation, it is a typical quantitative trait, and the improvement speed of traditional breeding method is slow. Although several key genes have been reported (such as NRT1.1B gene, OsAMT1 gene and OsGSr gene, etc.), they are involved in nitrate sensing, ammonium absorption and nitrogen assimilation process, respectively, but the key gene resources that can significantly enhance the ability of rice to resist low-nitrogen stress and maintain normal growth under low-nitrogen stress are still scarce. Under the condition of insufficient nitrogen supply, rice generally shows decreased biomass, reduced tiller number and smaller panicle, ultimately leading to significantly reduced yield. Therefore, mining new genes that can maintain the growth potential and yield potential of rice under low-nitrogen conditions has important application prospects for breeding new varieties with fertilizer-saving and strong stress resistance. SUMMARY

[0006] In view of the above problems, the purpose of the present application is to provide OsNER1 Application of a gene in regulating nitrogen utilization efficiency and yield of rice, in order to achieve the above purpose, the technical scheme adopted by the present application is: OsNER1 The nucleotide sequence of the gene is shown as SEQ ID NO. 1, and the gene OsNER1 The gene negatively regulates the nitrogen transport capacity of rice under low-nitrogen culture conditions.

[0007] Further, the gene OsNER1The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.2.

[0008] Furthermore, knock out the aforementioned OsNER1 Gene targets, obtained Osner1 mutant plants, Osner1 The mutant plants exhibit enhanced nitrogen transport capacity under low-nitrogen culture conditions.

[0009] Furthermore, compared to wild-type plants, the aforementioned Osner1 The aboveground height and biomass of mutant plants were significantly increased under low nitrogen culture conditions.

[0010] Furthermore, under low-nitrogen culture conditions, the Osner1 The aboveground and underground parts of the mutant plant 15 The nitrogen content was significantly higher in the aboveground and underground parts than in the wild-type plants. 15 The nitrogen content ratio is positively correlated with nitrogen transport capacity.

[0011] Furthermore, the rice is japonica rice, and the variety of japonica rice is Zhonghua 11.

[0012] A sort of OsNER1 Methods for constructing gene knockout vectors include: Get OsNER1 The gene sequence; The CRISPR design tools were used to design the... OsNER1 The gene sequences of the genes are identified and arranged to obtain candidate sgRNA target sites; The off-target sites of the predicted candidate sgRNA target sites are calculated to obtain target sgRNA-1 and target sgRNA-2; The target sgRNA-1 and target sgRNA-2 were knocked out respectively to obtain the knockout products; The knockout product was used to construct a vector to obtain the CRISPR-cas9 plasmid.

[0013] Furthermore, the sequences of target sgRNA-1 and target sgRNA-2 are as follows: sgRNA1: 5'-GCTGTGGGAGACGACCCAGA AGG-3'; sgRNA2: 5'-AGATCCTCTGAAGAATCGCG CGG -3'.

[0014] Compared with the prior art, the beneficial effects of the present invention include: OsNER1 The nucleotide sequence of the gene is shown in SEQ ID NO.1. OsNER1Genes negatively regulate the nitrogen transport capacity of rice under low-nitrogen culture conditions. OsNER1 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.2. CRISPR-Cas9 technology was used for... OsNER1 Gene editing, by knocking out rice OsNER1 Gene targets, obtained Osner1 Mutant plants. Experimental results showed that under low nitrogen conditions (0.2 mM NH3 concentration), compared with wild-type plants, Osner1 The mutant plants exhibited significantly increased aboveground height and biomass, effectively overcoming the growth inhibition caused by low nitrogen stress. Under low nitrogen conditions (0.2 mM NH4+), Osner1 The aboveground and underground parts of the mutant plant 15 The nitrogen content was significantly higher in the nitrogen-containing plants than in the wild-type plants, indicating that... OsNER1 The deletion of the gene enhances the plant's ability to absorb or transport nitrogen under low nitrogen conditions, thereby alleviating the inhibitory effect of low nitrogen stress on plant growth to some extent. OsNER1 This synergistic regulatory capacity of genes provides core gene resources for breeding new "fertilizer-saving and high-yield" rice varieties, which is of great significance to the sustainable development of agriculture. 34 NO3 Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0016] Figure 1 Rice knockout for the present invention OsNER1 Schematic diagram of gene targets; Figure 2 The wild-type plant (ZH11) of this invention and Osner1 mutant plants ( Osner1 -1) Statistical graph of aboveground height under different nitrogen concentration culture conditions; Figure 3 The wild-type plant (ZH11) of this invention and Osner1 mutant plants ( Osner1 -1) Biomass statistics under culture conditions with different nitrogen concentrations; Figure 4 The wild-type plant (ZH11) of this invention and Osner1 mutant plants ( Osner1 -2) Statistical graph of aboveground height under different nitrogen concentration culture conditions; Figure 5 The wild-type plant (ZH11) of this invention and Osner1 mutant plants (Osner1 -2) Biomass statistics under different nitrogen concentration culture conditions; Figure 6 The wild-type plant (ZH11) of this invention and Osner1 mutant plants ( Osner1 -1) Agronomic traits under culture conditions with different nitrogen concentrations; Figure 7 The wild-type plant (ZH11) of this invention and Osner1 mutant plants ( Osner1 -1) Aboveground and underground parts under different nitrogen concentration culture conditions 15 Statistical chart of nitrogen content ratio. Detailed Implementation

[0017] The following embodiments are used to illustrate the present invention, but are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of protection of the present invention. Unless otherwise specified, the experimental materials, reagents, instruments, etc., used in the embodiments of the present invention are commercially available; unless otherwise specified, all technical means in the embodiments of the present invention are conventional means well known to those skilled in the art.

[0018] Example 1 Rice OsNER1 Construction of gene knockout vector First obtain OsNER1 The gene sequence was obtained by searching for the gene number LOC_Os09g35000 using the National Rice Genome Annotation Database (https: / / ricedata.cn / gene / ). OsNER1 A gene's complete genomic DNA sequence, including exons, introns, and upstream and downstream regulatory regions. OsNER1 The nucleotide sequence of the gene is shown in SEQ ID NO.1. OsNER1 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.2.

[0019] Next, the knockout target was designed and the knockout vector was constructed. A suitable knockout target was designed using the CRISPR design tool, which can be found at http: / / cbi.hzau.edu.cn / CRISPR2 / . OsNER1The gene sequence of the gene is input into the CRISPR design tool, which generates a sgRNA candidate list according to key parameters such as on-target efficiency score, off-target effect prediction, target site location and genome characteristics, and selects suitable sgRNA from the sgRNA candidate list for knockout. The on-target efficiency score predicts the efficiency of the sgRNA guiding Cas9 to cut, and the highest score is selected in the on-target efficiency score. The off-target effect prediction tool scans the entire genome for other sites similar to the sgRNA sequence, and selects those sgRNAs with very low similarity to other positions in the genome in the off-target effect prediction to avoid cutting in the wrong place. The target site location mainly ensures that the target site is located within the coding sequence (CDS), and its PAM sequence (NGG) is located in the exon. The genome characteristics mainly avoid targeting repetitive sequence regions, SNP sites, etc. By using the CRISPR design tool to identify and arrange the gene sequence of the gene, candidate sgRNA target sites are obtained, and the off-target sites of each candidate sgRNA target site are calculated, thereby screening out two target sites sgRNA-1 and sgRNA-2. After knocking out the two target sites sgRNA-1 and sgRNA-2, the knockout product is obtained, and the knockout product is subjected to vector construction, and finally the CRISPR-cas9 plasmid is obtained, that is, the knockout vector of the gene. OsNER1 After the gene sequence of the gene is identified and arranged, candidate sgRNA target sites are obtained, and the off-target sites of each candidate sgRNA target site are calculated, thereby screening out two target sites sgRNA-1 and sgRNA-2. After knocking out the two target sites sgRNA-1 and sgRNA-2, the knockout product is obtained, and the knockout product is subjected to vector construction, and finally the CRISPR-cas9 plasmid is obtained, that is, the knockout vector of the gene. Osner1 .

[0020] Finally, mutant plants are obtained. Osner1 After the CRISPR-cas9 plasmid is subjected to plant genetic transformation, T0 generation plants are obtained, and the T0 generation plants are self-crossed to obtain T1 generation plants, and the T1 generation plants are self-crossed to obtain T2 generation plants, and the subsequent generation plants are cultivated and screened, and finally stable hereditary homozygous Osner1 mutant plants are obtained, including two homozygous Osner1 mutant plants, respectively named Osner1 -1 and OsNER1 -2.

[0021] Example 2 Rice Osner1 Gene knockout line seedling aboveground height determination Wild type plants (ZH11) and Osner1 mutant plants ( Osner1 -1 and Figure 2-2) Select 48 plump and normal seeds each, wash them 2-3 times with pure water to remove all impurities from the seed surface, then add 0.03% hydrogen peroxide to a depth of 1 cm and soak in the dark for 24 hours. On the third day after soaking, a white coleoptile will be visible emerging. Discard the waste liquid and wash the seeds 3-5 times with tap water. Once the radicle breaks through the husk, the seeds are ready for sowing. Fill the culture box with nutrient solution and place a 96-well plate on top. Select plump seeds with normal seed coat color and sow them in the 96-well plate. Set up two nitrogen nutrient conditions: low nitrogen (0.2 mM NH3) and high nitrogen (2 mM NH3), with 48 seedlings in each group. After sowing, place the seedlings in an artificial climate chamber (28℃, 12 / 12 photoperiod) for seedling growth. When the seedlings reach 15 days old, measure the above-ground height of the seedlings with a ruler. 4NO 34 NO3 The results are as follows Figure 4 and Osner1 As shown, under low nitrogen conditions (0.2 mM NH3), compared to wild-type plants, Osner1 mutant plants ( Osner1 -1 and Osner1 -2) The aboveground height of the seedlings increased significantly, indicating Osner1 The mutant plants effectively alleviated growth inhibition caused by nitrogen deficiency and enhanced their adaptability to infertile soils. Under high-nitrogen conditions (2 mM NH3 concentration), compared with wild-type plants, Osner1 mutant plants ( Osner1 -1) The aboveground height of the seedlings increased significantly, indicating Osner1 mutant plants ( OsNER1 -1) It still has the potential to increase production under high nitrogen conditions. 4NO 34 NO3 Example 3 Rice Osner1 Biomass determination of gene knockout line seedlings Wild-type plants (ZH11) and Osner1 mutant plants ( Osner1 -1 and Figure 3-2) Select 48 plump and normal seeds each, wash the seeds 2-3 times with pure water to remove all impurities from the seed surface, then add 0.03% hydrogen peroxide to a depth of 1 cm to soak in the dark for 24 hours. On the third day after soaking, a white coleoptile will be visible emerging. Discard the waste liquid and wash the seeds 3-5 times with tap water. Once the radicle breaks through the glume, the seeds are ready for sowing. Fill the culture box with nutrient solution and place a 96-well plate on top. Select plump seeds with normal seed coat color and sow them in the 96-well plate. Set up two nitrogen nutrient conditions: low nitrogen (0.2 mM NH3) and high nitrogen (2 mM NH3), with 48 seedlings in each group. After sowing, place the seedlings in an artificial climate chamber (28℃, 12 / 12 photocycle) for seedling cultivation. When the seedlings reach 15 days old, take 30 seedlings, place them in an envelope, and put them in a 42℃ oven for 2-3 days until the seedlings are completely dehydrated. Then, weigh the dry weight of each seedling using an analytical balance. 34 NO3 The results are as follows Figure 5 and Osner1 As shown, under low nitrogen conditions (0.2 mM NH3), compared to wild-type plants, Osner1 mutant plants ( Osner1 -1 and Osner1 -2) The seedling biomass of all seedlings increased significantly, indicating Osner1 mutant plants ( Osner1 -1 and Osner1 -2) Effectively overcame the inhibitory effect of low nitrogen stress on plant growth. Under high nitrogen conditions (2 mM NH3 concentration), compared with wild-type plants, Osner1 mutant plants ( Osner1 The biomass of -1) increased significantly, indicating Osner1 mutant plants ( OsNER1 -1) It still has the potential to increase production under high nitrogen conditions. 4NO 34 NO3 Example 4 Rice Osner1 Determination of nitrogen uptake capacity in gene knockout seedlings Wild-type plants (ZH11) and Osner1 mutant plants ( Figure 7Select several plump and normal seeds (-1) and wash them 2-3 times with pure water to remove all impurities from the seed surface. Then, add 0.03% hydrogen peroxide to a depth of 1 cm above the seeds and soak them in the dark for 24 hours. On the third day after soaking, a white, distinct coleoptile will emerge. Discard the waste liquid and wash the seeds 3-5 times with tap water. Transfer the germinated seeds to a microgrid with multiple small grids and place them in an artificial climate chamber (28℃, 12 / 12 photoperiod). Change the water regularly every day. After about 2-3 days, when the seeds have grown roots and leaves, select seedlings with consistent above-ground and underground growth and transfer them to 96-well plates. Place the plates in culture boxes filled with nutrient solution. Set up two nitrogen nutrient conditions: low nitrogen (0.2 mM NH4+) and high nitrogen (2 mM NH4+), with 48 seedlings in each group. After sowing, place the seedlings in an artificial climate chamber (28℃, 12 / 12 photoperiod) for seedling cultivation. When the seedlings reached 15 days old, they were subjected to nitrogen starvation treatment, i.e., KCl was used instead of NH4NO. After 3 days of treatment, they were transferred to a low-nitrogen environment with a nitrogen concentration of 0.2 mM. 15 NH4 15 NO and high nitrogen (i.e., nitrogen concentration of 2 mM) 15 NH4 15 In NO, after treatment for 30 min, the seedlings were immediately rinsed in 0.1 mM CaSO4 solution for 1 min. The roots and stems were then cut open at the junction with scissors, and the above-ground and underground parts were placed separately into kraft paper bags and dried in a 65℃ oven for 3 days. Each group consisted of 3 seedlings, for a total of 4 replicates. The dry weight of the above-ground and underground parts in each group was weighed and recorded. The parts were then shredded into powder, collected, and passed through a 100-mesh sieve for testing. The nitrogen uptake capacity of the seedlings was calculated based on the measured dry weight and the detected concentration. 34 NO 3333 The results are as follows Osner1 As shown, under low nitrogen conditions, i.e., a nitrogen concentration of 0.2 mM NH, Osner1 mutant plants ( OsNER1 -1) The above-ground part is greater than the underground part 15 The N content ratio was significantly higher than that of the wild-type plant (ZH11), indicating that... OsNER1 The deletion of the gene enhanced the plant's nitrogen uptake or translocation capacity under low nitrogen conditions, thus alleviating the inhibitory effect of low nitrogen stress on plant growth to some extent. Further mechanistic studies have shown that... OsNER1 Genes may influence the germination and elongation of tillers by regulating nitrogen signal transduction pathways, thereby maintaining a high tillering rate under nitrogen-limited conditions. ​ The loss-of-function gene enhances stress resistance under low nitrogen conditions while retaining yield-increasing potential under high nitrogen conditions, demonstrating its regulatory advantage in balancing the traits of "stress resistance" and "high yield" under different nitrogen levels. This provides new gene targets and design ideas for future nitrogen-efficient rice breeding. It can be understood that the above embodiments only express the preferred embodiments of the present application, which are described in a more specific and detailed manner, but cannot be understood as a limitation to the patent scope of the present application; it should be pointed out that for ordinary skilled in the art, the above technical features can be freely combined without departing from the concept of the present application, and several modifications and improvements can be made, which all belong to the protection scope of the present application; therefore, any equivalent transformation and modification made to the patent claim scope of the present application shall belong to the coverage of the patent claim of the present application.

Claims

1. OsNER1 The application of genes in regulating nitrogen use efficiency and yield in rice, characterized in that, OsNER1 The nucleotide sequence of the gene is shown as SEQ ID NO. 1, and the OsNER1 The gene negatively regulates the nitrogen transport capacity of rice under low nitrogen culture conditions.

2. Use according to claim 1, characterized in that, The OsNER1 The amino acid sequence of the protein encoded by the gene is shown as SEQ ID NO.

2.

3. Use according to claim 1, characterized in that, knocking out the target of the gene, obtaining OsNER1 mutant plants, Osner1 mutant plants, Osner1 The nitrogen transport capacity of the mutant plants is enhanced under low nitrogen culture conditions.

4. Use according to claim 3, characterized in that, The mutant plants have increased height and biomass in the aerial part under low nitrogen culture conditions compared to wild type plants. Osner1 The mutant plants have increased height and biomass in the aerial part under low nitrogen culture conditions compared to wild type plants.

5. Use according to claim 3, characterized in that, In the low nitrogen culture condition, the aboveground part and the underground part of the mutant plant have a significantly higher N content than the wild type plant Osner1 The N content of the aboveground part and the underground part of the mutant plant is positively correlated with the nitrogen transport capacity 15 The N content of the aboveground part and the underground part of the mutant plant is positively correlated with the nitrogen transport capacity 15 The N content of the aboveground part and the underground part of the mutant plant is positively correlated with the nitrogen transport capacity 6. Use according to any one of claims 1 to 5, characterized in that, The rice is japonica, and the variety of the japonica is Zhonghua 11.

7. A kind OsNER1 A method for constructing a gene knockout vector, characterized in that, comprising: Acquisition OsNER1 Gene sequence of a gene; The CRISPR design tool is used to recognize and arrange the gene sequences of the genes to obtain candidate sgRNA target sites. OsNER1 The CRISPR design tool is used to recognize and arrange the gene sequences of the genes to obtain candidate sgRNA target sites. OsNER1 The off-target sites of the candidate sgRNA target sites are calculated and prediction, to obtain target sgRNA-1 and target sgRNA-2; The target sgRNA-1 and target sgRNA-2 are respectively subjected to knockout, to obtain post-knockout products; 8. The method of claim 7, wherein, The post-knockout products are subjected to vector construction, to obtain CRISPR-cas9 plasmids. The sequences of the target sgRNA-1 and target sgRNA-2 are: sgRNA1: 5'-GCTGTGGGAGACGACCCAGA AGG-3'; sgRNA2: 5'-AGATCCTCTGAAGAATCGCG CGG -3'.

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