OsYABBY4 gene for regulating and controlling nitrogen deficiency stress resistance of rice and application of OsYABBY4 gene
By overexpressing the OsYABBY4 gene in rice, the rice's tolerance to nitrogen deficiency stress was regulated, solving the problem of low nitrogen fertilizer utilization in rice and improving its growth performance under nitrogen-deficient conditions.
Patent Information
- Application Number
- CN202511830431.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-06
- Publication Date
- 2026-01-23
AI Technical Summary
The current nitrogen fertilizer utilization rate in rice is low, leading to increased production costs and ecological environmental pressure. Furthermore, there are few nitrogen-efficient genes that have been successfully applied in rice.
By overexpressing the OsYABBY4 gene in rice, the nitrogen deficiency tolerance of rice can be regulated, thereby improving the growth performance of rice under nitrogen deficiency conditions.
It significantly increases plant height and yield per plant in rice under nitrogen deficiency stress, and enhances the rice's tolerance to nitrogen deficiency stress.
Smart Images
Figure FT_1 
Figure FT_2 
Figure FT_3
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the field of biotechnology, in particular to a gene for regulating the nitrogen deficiency stress tolerance of rice and its application. OsYABBY4 BACKGROUND
[0002] Since the 1960s, the first "green revolution" characterized by semi-dwarf breeding has greatly improved crop yield, but has also brought the problem of reduced nitrogen use efficiency. Carrying "green genes", crops have a high accumulation of DELLA protein in their bodies, resulting in reduced nitrogen response and utilization efficiency. The average nitrogen fertilizer use efficiency of rice in China is only 30%~35%, which is relatively low. In order to increase yield, a large amount of nitrogen fertilizer must be applied, which not only increases the production cost of farmers, but also causes great pressure on the ecological environment, so cultivating nitrogen-efficient crop varieties has become the key to solving this problem.
[0003] In the field of rice, Chinese scientists have proposed the concept of "green super rice", which reduces the use of nitrogen fertilizer while ensuring yield. There are a large number of transport proteins in plants that participate in the absorption and utilization of nitrogen, which plays an important role in the formation of yield and quality of rice. At present, a large number of genes related to nitrogen transport and utilization have been mined in rice, such as nitrate transporters (NRT), ammonium transporters (AMT), NIN-like protein (NLP) family transcription factors, etc., which play an important role in the absorption, transport and assimilation of nitrogen. For example, NRT proteins are mainly responsible for the transport and utilization of nitrate nitrogen; AMT proteins are mainly responsible for the transport and utilization of ammonium nitrogen; NLR family genes act as transcription factors in the process of regulating downstream genes; in addition, OsGRF4 is a positive regulator of plant carbon-nitrogen metabolism, which can promote nitrogen absorption, assimilation and transport, and thus promote plant growth and development. The plant nitrogen regulation network is relatively complex, although some nitrogen-efficient related genes have been identified, but few have been successfully applied to practical production, so mining more related genes, creating high-quality germplasm resources, and applying them to practical production are still the focus of attention. SUMMARY
[0004] The present application relates to the field of biotechnology, in particular to a gene for regulating the nitrogen deficiency stress tolerance of rice and its application. OsYABBY4
[0005] To achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows: OsYABBY4 The application of genes in regulating the nitrogen deficiency stress tolerance of rice, the aforementioned OsYABBY4 The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO.2; Furthermore, overexpression in rice OsYABBY4 Genes can improve rice's tolerance to nitrogen deficiency stress.
[0006] A method for regulating the nitrogen deficiency stress tolerance of rice, the method comprising regulating the nitrogen deficiency stress tolerance of rice. OsYABBY4 The steps for gene expression levels; OsYABBY4 The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO.2; Furthermore, the medium-lowering concentration of rice was increased. OsYABBY4 Gene expression can enhance rice's tolerance to nitrogen deficiency stress.
[0007] OsYABBY4 The application of the gene in breeding transgenic rice with improved nitrogen-deficiency stress resistance, the OsYABBY4 The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO.2; Furthermore, OsYABBY4 The gene was overexpressed in the recipient rice to obtain transgenic rice; the transgenic rice had a higher tolerance to nitrogen deficiency stress than the recipient rice.
[0008] A method for breeding transgenic rice with enhanced nitrogen-deficiency stress resistance, OsYABBY4 The gene was overexpressed in recipient rice to obtain transgenic rice; the transgenic rice showed higher tolerance to nitrogen deficiency stress than the recipient rice; OsYABBY4 The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO.2.
[0009] OsYABBY4 The application of genes in promoting rice growth under nitrogen deficiency stress, the aforementioned OsYABBY4 The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO.2; Furthermore, the promotion of rice growth under nitrogen deficiency stress refers to promoting increased plant height and / or increased yield per plant under nitrogen deficiency stress; the yield per plant refers to the grain weight per plant.
[0010] A method to promote rice growth under nitrogen deficiency stress, which involves... OsYABBY4The gene was overexpressed in the recipient rice to obtain transgenic rice; the transgenic rice exhibited higher plant height and / or yield per plant under nitrogen deficiency stress than the recipient rice; OsYABBY4 The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO.2; the yield per plant is the grain weight per plant.
[0011] The significant advantages of this invention are: This invention discovers a new type of rice OsYABBY4 The gene is involved in regulating the ability to tolerate nitrogen deficiency stress, which is of great significance to the research on "low nitrogen and high efficiency" in rice, and has certain application value for high-quality rice breeding and to help the new round of "green revolution". Attached Figure Description
[0012] Figure 1 : OsYABBY4 Gene structure and OsYABBY4-OE Results of gene expression analysis of overexpression lines.
[0013] Figure 2 : OsYABBY4 Observation and statistical analysis of plant height phenotype in overexpressing rice after 10 days of culture in nitrogen-deficient nutrient solution.
[0014] Figure 3 : OsYABBY4 Observation and statistical analysis of plant height phenotype in overexpressing rice cultured under low nitrogen conditions until maturity.
[0015] Figure 4 : OsYABBY4 Observation and statistical analysis of yield phenotype of single plant in rice with gene overexpression under low nitrogen conditions.
[0016] Note:**, p <0.01. Detailed Implementation
[0017] To illustrate the technical content, objectives, and effects of this invention in detail, the following description is provided in conjunction with the embodiments and accompanying drawings. Unless otherwise specified, the experimental methods used in the embodiments are conventional methods; and unless otherwise specified, the materials and reagents used are commercially available.
[0018] Rice in this invention OsYABBY4 The CDS sequence of the gene is shown in SEQ ID NO.1, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO.2.
[0019] The primer sequences used in this invention are as follows:
[0020] The Yoshida rice nutrient solution used in this invention was purchased from Beijing Kulaibo Technology Co., Ltd., product number NS1040; the nitrogen-deficient Yoshida rice nutrient solution used in this invention was purchased from Beijing Kulaibo Technology Co., Ltd., product number NS1040-N.
[0021] Example 1: OsYABBY4 Construction of gene overexpression rice (1) Using cDNA from spikelet tissue at maturity of the japonica rice variety Zhonghua 11 as a template, primers YABBY4-PENTR-F and YABBY4-PENTR-R were used to target cDNA from the spikelet tissue of the japonica rice variety Zhonghua 11. OsYABBY4 The CDS sequence of the gene was amplified by PCR. (2) The PCR product obtained in (1) was ligated to the intermediate vector pENTR™ / D-TOPO (Invitrogen) using Gateway technology to obtain the recombinant vector pENTR™ / D-TOPO- OsYABBY4 After sequencing verification, the vector was subjected to LR recombination with the final vector pGWB505 to obtain the recombinant vector pGWB505- for overexpression. OsYABBY4 The recombinant vector pGWB505- OsYABBY4 E. coli DH5α competent cells were transformed using the heat shock method. Positive clones were selected and sent for sequencing. The recombinant vector, verified by sequencing, was transformed into Agrobacterium tumefaciens EHA105 using the electroporation method. After the transformants were verified to be correct, callus tissue of the japonica rice variety Zhonghua 11 was transformed. Through a series of processes including screening, differentiation, and rooting, the desired recombinant vector was obtained. OsYABBY4 Rice plants overexpressing the gene are denoted as OsYABBY4-OE .
[0022] In a smart greenhouse (temperature 26~28℃, air humidity 50%, 14h light / 10h darkness), wild-type medium-flowered 11, OsYABBY4 After germination, rice seeds overexpressing the gene were cultured in Yoshida rice nutrient solution (nitrogen concentration 40 ppmol / L), with the solution changed every 2 days. After 7 days of culture, leaves were harvested and primed using primers YABBY4-qPCR-F and YABBY4-qPCR-R to detect wild-type Zhonghua 11 and... OsYABBY4 In rice with overexpressed genes OsYABBY4 Gene expression levels are detected using quantitative PCR. For example... Figure 1 As shown, OsYABBY4 Genes in OsYABBY4 The gene overexpression level in rice was significantly higher than that in wild-type Zhonghua 11.
[0023] In a smart greenhouse (temperature 26~28℃, air humidity 50%, 14h light / 10h darkness), wild-type medium-flowered 11, OsYABBY4After the seed germination of the gene overexpression rice, the rice seedlings are placed in Yoshida rice nutrient solution (nitrogen concentration 40 ppmol / L) and nitrogen-free Yoshida rice nutrient solution (nitrogen concentration 0 ppmol / L) respectively, liquid is changed every 2 days, and after 10 days of culture, the plant height of the rice seedlings is observed and counted. As shown in Figure 2 , the rice planted in the Yoshida rice nutrient solution, OsYABBY4 the plant height of the overexpression strain is basically the same as that of the wild type; and the rice planted in the nitrogen-free Yoshida rice nutrient solution, OsYABBY4 the plant height of the overexpression strain is significantly increased compared with the wild type.
[0024] The experiment is carried out in an intelligent greenhouse (temperature 26-28°C, air humidity 50%, 14h light / 10h darkness). In order to ensure the uniformity of the soil background nutrients and physical and chemical properties, two planting pools with completely consistent soil conditions are selected, and wild type Zhonghua 11 and OsYABBY4 gene overexpression rice are planted respectively. On this basis, two nitrogen supply treatments are set: one pool adopts normal fertilization scheme (normal nitrogen level, Normal group), that is, nitrogen fertilizer is applied at a dosage of 135 kg / hm 2 of pure nitrogen, and is applied in several times according to the ratio of base fertilizer: tiller fertilizer: ear fertilizer = 5:3:2; the other pool does not apply additional fertilizer, and only relies on the soil itself to build a low nitrogen stress environment (low nitrogen, LN group). After the rice plants are cultured to complete maturity, systematic phenotype observation and related agronomic trait statistical analysis are carried out. As shown in Figure 3 , under the condition of normal nitrogen level, OsYABBY4 the plant height of the overexpression strain has no significant difference compared with the wild type Zhonghua 11; under the condition of low nitrogen, OsYABBY4 the plant height of the overexpression strain is significantly increased compared with the wild type Zhonghua 11. As shown in Figure 4 , taking single plant grain weight as the yield representation index, under the condition of normal nitrogen level, OsYABBY4 the single plant yield of the overexpression strain has no significant difference compared with the wild type Zhonghua 11; under the condition of low nitrogen, OsYABBY4 the single plant yield of the overexpression strain is significantly increased compared with the wild type Zhonghua 11.
[0025] This study proves OsYABBY4 that the gene participates in the regulation of the nitrogen deficiency tolerance of rice, and has certain significance for the regulation of rice “low nitrogen efficiency” and rice quality breeding.
[0026] The above only describes the preferred embodiments of the present application, and any changes and modifications made within the scope of the patent application of the present application shall be included in the scope of the present application.
Claims
1. OsYABBY4 The application of genes in regulating the ability of rice to tolerate nitrogen deficiency stress is characterized by: The OsYABBY4 The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO.
2.
2. The application according to claim 1, characterized in that: Overexpression in rice OsYABBY4 Genes that enhance rice's tolerance to nitrogen deficiency stress.
3. A method for regulating the tolerance of rice to nitrogen deficiency stress, characterized in that: The method includes regulating the rice... OsYABBY4 The steps for gene expression levels; OsYABBY4 The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO.
2.
4. The method according to claim 3, characterized in that: Increase rice OsYABBY4 Gene expression to enhance rice's tolerance to nitrogen deficiency stress.
5. OsYABBY4 The application of genes in breeding transgenic rice with enhanced nitrogen-deficiency stress is characterized by: The OsYABBY4 The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO.
2.
6. The application according to claim 5, characterized in that: Will OsYABBY4 The gene was overexpressed in the recipient rice to obtain transgenic rice; the transgenic rice had a higher tolerance to nitrogen deficiency stress than the recipient rice.
7. A method for cultivating transgenic rice with enhanced nitrogen-deficiency stress resistance, characterized in that: Will OsYABBY4 The gene was overexpressed in the recipient rice to obtain transgenic rice; the transgenic rice had a higher tolerance to nitrogen deficiency stress than the recipient rice. The OsYABBY4 The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO.
2.
8. OsYABBY4 The application of genes in promoting rice growth under nitrogen deficiency stress is characterized by: The OsYABBY4 The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO.
2.
9. The application according to claim 8, characterized in that: The promotion of rice growth under nitrogen deficiency stress refers to promoting increased plant height and / or increased yield per plant under nitrogen deficiency stress; the yield per plant refers to the grain weight per plant.
10. A method for promoting rice growth under nitrogen deficiency stress, characterized in that: Will OsYABBY4 The gene was overexpressed in the recipient rice to obtain transgenic rice; the transgenic rice had a higher plant height and / or yield per plant than the recipient rice under nitrogen deficiency stress. The OsYABBY4 The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO.2; the yield per plant is the grain weight per plant.