Application of OsBTD1 gene in regulation and control of nitrogen utilization efficiency of rice

Abstract

The invention provides application of an OsBTD1 gene in regulation and control of rice nitrogen utilization efficiency, the nucleotide sequence of the OsBTD1 gene is shown as SEQ ID NO.1, and the OsBTD1 gene negatively regulates and controls the absorption rate of rice to nitrogen. According to the present invention, the target of the OsBTD1 gene is knocked out to obtain the osbtd1 mutant plant, the height of the overground part of the osbtd1 mutant plant under the low nitrogen stress condition is significantly higher than the height of the wild type plant, and the osbtd1 mutant plant effectively overcomes the inhibition of the low nitrogen stress on the plant growth; the height of the overground part of the osbtd1 mutant plant under the high-nitrogen condition is obviously higher than that of a wild type plant, so that the osbtd1 mutant plant still has yield increasing potential under the high-nitrogen condition. The cooperative regulation capability of the OsBTD1 gene provides a core gene resource for cultivating a new variety of fertilizer-saving high-yield rice, and has great significance for agricultural sustainable development.

Description

Technical Field

[0001] This invention belongs to the field of plant breeding and biological gene technology, specifically involving OsBTD1 Application of genes in regulating nitrogen use efficiency in rice. Background Technology

[0003] Food security is a crucial cornerstone of national strategy. As a major staple crop, rice's nitrogen use efficiency directly impacts production efficiency and the ecological environment. Nitrogen is a key mineral nutrient essential for rice growth and development, participating in the synthesis of various vital substances such as proteins and chlorophyll, and playing a decisive role in yield formation. However, current rice production commonly suffers from excessive nitrogen fertilizer application and low utilization rates; approximately 60%–70% of nitrogen is not absorbed by crops and is lost to the environment, increasing production costs and causing serious environmental pollution. Therefore, identifying key genes for regulating nitrogen-efficient use and cultivating "fertilizer-saving and high-yield" rice varieties are urgent needs for achieving sustainable agricultural development.

[0004] In breeding practice, selecting nitrogen-efficient varieties is considered the fundamental way to improve nitrogen fertilizer utilization. However, because nitrogen absorption, translocation, and assimilation involve the coordinated regulation of multiple genes and are complex quantitative traits, traditional breeding methods have made slow progress. Although some key genes (such as...) NRT1.1B , OsAMT1 , OsGSr (e.g.,) have been reported to participate in nitrogen sensing and assimilation processes, but key genes that can effectively maintain plant vigor and ensure yield under low nitrogen stress are still relatively lacking. Summary of the Invention

[0005] To address the above problems, the purpose of this invention is to provide... OsBTD1 To achieve the above objectives, the technical solution adopted in this invention is as follows: [This section discusses the application of genes in regulating nitrogen use efficiency in rice.] OsBTD1 The nucleotide sequence of the gene is shown in SEQ ID NO.1. OsBTD1 Genes negatively regulate the rate of nitrogen uptake in rice.

[0006] Furthermore, the aforementioned OsBTD1 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.2.

[0007] Furthermore, knock out the aforementioned OsBTD1 Gene targets, obtained osbtd1 mutant plants, the osbtd1 The mutant plants exhibit enhanced resistance to low nitrogen conditions, including mitigation of growth inhibition caused by nitrogen deficiency and enhanced plant adaptability to poor environments.

[0008] Furthermore, the aforementioned osbtd1 The aboveground height of the mutant plants under low nitrogen stress was significantly greater than that of the wild-type plants. osbtd1 The aboveground height of mutant plants under high nitrogen conditions was significantly greater than that of wild-type plants.

[0009] Furthermore, the nitrogen concentration under the low-nitrogen condition is 0.2 mM NH4NO3, and the nitrogen concentration under the high-nitrogen condition is 2 mM NH4NO3.

[0010] Furthermore, the rice variety is Zhonghua 11.

[0011] A method for constructing a knockout vector for knocking out [a specific substance] in rice. OsBTD1 The method includes: targeting a gene. Get OsBTD1 The gene sequence; Based on the gene sequence, target sequences were designed using CRISPR tools; The CRISPR-cas9 plasmid was obtained by knocking out the sgRNA1 and sgRNA2 dual target sites in the target sequence.

[0012] Furthermore, the design of target sequences using CRISPR tools includes: Identify and arrange appropriate target sites to obtain the specified target; Calculate and predict the off-target sites for each of the specified targets.

[0013] Furthermore, the target sequence is as follows: sgRNA1: 5'-CCAGAACTTGCGCCGGTACG CGG-3'; SgRNA2: 5'-GCACGAGCTCCTCGTCGTAG AGG-3'.

[0014] Compared with the prior art, the beneficial effects of the present invention include: OsBTD1 The nucleotide sequence of the gene is shown in SEQ ID NO.1. OsBTD1 Genes negatively regulate the rate of nitrogen uptake in rice. OsBTD1 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.2. CRISPR-Cas9 technology was used to analyze the protein. OsBTD1 Gene editing, through gene knockout OsBTD1 Gene targets, obtained osbtd1 mutant plants, osbtd1 The aboveground height of the mutant plants was significantly greater than that of the wild-type plants under low nitrogen stress. osbtd1The mutant plants effectively overcame the inhibition of plant growth caused by low nitrogen stress and enhanced the plants' nitrogen utilization and adaptability in barren environments. osbtd1 The aboveground height of the mutant plants under high nitrogen conditions was significantly greater than that of the wild-type plants, indicating that... osbtd1 The mutant plants still exhibited yield-increasing potential under high-nitrogen conditions, demonstrating a significant advantage in synergistically regulating low-nitrogen tolerance and high-yield traits under different nitrogen levels. This invention is the first to discover and confirm this. OsBTD1 The dual function of genes in regulating nitrogen response in rice OsBTD1 This synergistic regulatory capacity of genes provides core gene resources for breeding fertilizer-saving and high-yield rice varieties, which is of great significance to the sustainable development of agriculture. 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 variety knockout for the present invention OsBTD1 Schematic diagram of gene targets; Figure 2 The wild-type plant (ZH11) of this invention and osbtd1 Image showing the aboveground height of mutant seedlings; Figure 3 The wild-type plant (ZH11) of this invention and osbtd1 Agronomic trait diagram of the aboveground height of mutant seedlings. 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 OsBTD1 Construction of gene knockout vector rice OsBTD1 The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the amino acid sequence of the protein encoded by the rice OsBTD1 gene is shown in SEQ ID NO.2.

[0019] The gene sequence was obtained by searching for gene number LOC_Os01g55650 in the National Rice Data Center (https: / / ricedata.cn / gene / ), a rice genome annotation database.

[0020] Using the CRISPR design tool (http: / / cbi.hzau.edu.cn / CRISPR2 / ), a specialized bioinformatics software or online platform, the core function of which is to help researchers design highly efficient and specific guide RNAs (gRNA / sgRNA) for CRISPR gene editing experiments (especially the CRISPR-Cas9 system). First, input... OsBTD1 The gene sequence is obtained, and then suitable target sites are identified and arranged to obtain the specified targets. The off-target sites for each specified target are calculated and predicted. The CRISPR design tool lists all possible gRNAs and provides two core scores: efficiency score and specificity score. The efficiency score predicts the on-target cleavage efficiency, and the higher the score, the better. The specificity score predicts the off-target effect, and the lower the score, the better. Finally, the sgRNA1 single target is selected for knockout, and the CRISPR-cas9 plasmid is constructed.

[0021] The target sequence is: sgRNA1: 5'-CCAGAACTTGCGCCGGTACG CGG-3'; sgRNA2: 5'-GCACGAGCTCCTCGTCGTAG AGG-3'.

[0022] Example 2 Rice OsBTD1 Measurement of aboveground height of gene knockout seedlings Wild-type plants (ZH11) and osbtd1 Forty-eight plump and tall seeds from each mutant plant were washed 2-3 times with pure water to remove all impurities from the seed surface. Then, 0.03% hydrogen peroxide was added until the seeds were submerged 1 cm, and the seeds were soaked in the dark for 24 hours. On the third day after soaking, a distinct white coleoptile was visible emerging. The waste liquid was discarded, and the seeds were rinsed 3-5 times with tap water. Once the radicle broke through the husk, the seeds were ready for sowing. A 96-well plate was placed on top of a culture box filled with nutrient solution. Plump seeds with a bright seed coat color were selected and sown in the 96-well plate. Two nitrogen nutrient conditions were set up: low nitrogen (0.2 mM NH4+) and high nitrogen (2 mM NH4+), with 48 seedlings in each group. After sowing, the seedlings were placed in an artificial climate chamber (28℃, 12 / 12 photoperiod) for aging. The above-ground height of the seedlings was measured with a ruler when the seedlings reached 15 days of age. 34 NO3 The results are as follows Figure 2 and Figure 3 As shown, Figure 2 Reflects wild-type plants and osbtd1 Numerical quantitative differences in mutant plants Figure 3 Reflects wild-type plants and osbtd1 Qualitative differences in agronomic traits in mutant plants. Under low-nitrogen conditions (0.2 mM NH3), compared to wild-type plants (ZH11), osbtd1 The aboveground height of the mutant plants was significantly increased, while the average aboveground height of the wild-type plants was less than 30 cm. osbtd1 The average aboveground height of the mutant plants was greater than 30 cm and less than 40 cm, with p-values ​​less than 0.001 for both. Figure 3 It can be seen that, in terms of agronomic traits, under low nitrogen conditions, i.e., LN conditions, osbtd1 The aboveground height of the mutant plants was greater than that of the wild-type plants. These results demonstrate... osbtd1 The mutant plants effectively overcame the growth inhibition caused by low nitrogen stress and exhibited stronger adaptability to low nitrogen environments. Under high nitrogen conditions (2 mM NH3 concentration), compared to wild-type plants (ZH11), osbtd1 The aboveground height of the mutant plants was significantly increased, while the average aboveground height of the wild-type plants was less than 40 cm. osbtd1 The mutant plants had an average aboveground height greater than 40 cm, and the p-value was 0.001296. Figure 3 It can be seen that, in terms of agronomic traits, under high nitrogen conditions, i.e., HN conditions, osbtd1 The aboveground height of the mutant plants was greater than that of the wild-type plants. These results demonstrate... osbtd1 The mutant plants had a higher nitrogen utilization capacity than the wild-type plants under high nitrogen conditions, and osbtd1 The mutant plants still showed potential for increased yield under high nitrogen conditions. 4NO3 It is understood that the above embodiments only illustrate preferred embodiments of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can freely combine the above technical features without departing from the concept of the present invention, and can also make several modifications and improvements, all of which fall within the protection scope of the present invention. Therefore, all equivalent transformations and modifications made with respect to the scope of the claims of the present invention should fall within the scope of the claims of the present invention.

Claims

1. OsBTD1 The application of genes in regulating nitrogen use efficiency in rice is characterized by, OsBTD1 The nucleotide sequence of the gene is shown in SEQ ID NO.

1. OsBTD1 Genes negatively regulate the rate of nitrogen uptake in rice.

2. The application according to claim 1, characterized in that, The OsBTD1 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.

2.

3. The application according to claim 1, characterized in that, Knock out the OsBTD1 Gene targets, obtained osbtd1 mutant plants, the osbtd1 The mutant plants exhibit enhanced resistance to low nitrogen conditions, including mitigation of growth inhibition caused by nitrogen deficiency and enhanced plant adaptability to poor environments.

4. The application according to claim 3, characterized in that, The osbtd1 The mutant plants, under low nitrogen conditions, had greater aboveground height than the wild-type plants. osbtd1 The aboveground height of mutant plants under high nitrogen conditions was greater than that of wild-type plants.

5. The application according to claim 4, characterized in that, The nitrogen concentration under the low nitrogen condition is 0.2 mM NH4NO3, and the nitrogen concentration under the high nitrogen condition is 2 mM NH4NO3.

6. The application according to any one of claims 1-5, characterized in that, The rice variety mentioned is Zhonghua 11.

7. A method for constructing a knockout vector, characterized in that, Used for knocking out in rice as described in any one of claims 1-6 OsBTD1 The method includes: targeting a gene. Get OsBTD1 The gene sequence; Based on the gene sequence, target sequences were designed using CRISPR tools; The CRISPR-cas9 plasmid was obtained by knocking out the sgRNA1 and sgRNA2 dual target sites in the target sequence.

8. The construction method according to claim 7, characterized in that, The design of target sequences using CRISPR tools includes: Identify and arrange appropriate target sites to obtain the specified target; Calculate and predict the off-target sites for each of the specified targets.

9. The construction method according to claim 7, characterized in that, The target sequence is: sgRNA1: 5'-CCAGAACTTGCGCCGGTACG CGG-3'; sgRNA2: 5'-GCACGAGCTCCTCGTCGTAG AGG-3'.

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