Application of rice nitrogen utilization gene NU3 in regulating rice growth

Abstract

This invention discloses the application of the rice nitrogen utilization gene NU3 in regulating rice growth, belonging to the field of plant genetic engineering technology. The nucleotide sequence of the rice nitrogen utilization gene NU3 described in this invention is shown in SEQ ID NO.1; the regulation of rice growth refers to regulating rice nitrogen utilization rate, rice seed quality, and seed protein content. This invention identified the expression pattern of gene NU3 in rice and obtained its loss-of-function mutant using gene editing technology. This loss-of-function mutant resulted in reduced seedling growth, significantly decreased thousand-grain weight, decreased seed protein content, and decreased nitrogen utilization rate. This invention provides new gene resources and a research foundation for rice nitrogen metabolism research, growth regulation mechanism research, and seed quality regulation.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, and in particular to the application of the rice nitrogen utilization gene NU3 in regulating rice growth. Background Technology

[0002] Nitrogen is an essential nutrient element for the growth and development of crops such as rice, playing a crucial role in biomass accumulation and yield formation. In modern agricultural production, chemical nitrogen fertilizers are typically applied to meet the nitrogen requirements of crops in order to increase yield. However, excessive use of chemical nitrogen fertilizers not only increases production costs but also causes serious environmental problems, such as soil acidification, water eutrophication, and greenhouse gas emissions. Therefore, exploring genes and their regulatory mechanisms that improve nitrogen use efficiency in rice is of great significance for achieving sustainable agricultural development.

[0003] In recent years, with the development of genomics and molecular biology techniques, scientists have gained a deeper understanding of the molecular mechanisms of nitrogen absorption, transport, and metabolism in rice. Some studies have shown that genes related to nitrogen use efficiency in rice can significantly improve crop nitrogen use efficiency by regulating nitrogen absorption, allocation, and metabolic processes. The discovery of these genes provides new molecular targets for crop variety improvement and a theoretical basis for reducing the application of chemical nitrogen fertilizers. However, functional studies of most known nitrogen-regulating genes are still in their early stages, and the complexity of their regulatory mechanisms has not been fully revealed, especially regarding gene expression regulation and signal transduction pathways under nitrogen stress conditions, many mysteries remain. Therefore, discovering new nitrogen-regulating genes and their roles in rice, laying the foundation for breeding new rice varieties with high nitrogen use efficiency, is currently one of the research hotspots in the fields of agriculture and biotechnology.

[0004] Therefore, in-depth research on the function of nitrogen utilization-related genes and their role in nitrogen utilization in rice, and its application to rice genetic improvement, is of great significance for achieving efficient and green agriculture. Summary of the Invention

[0005] The purpose of this invention is to provide the application of the rice nitrogen utilization gene NU3 in regulating rice growth, thereby addressing the problems existing in the prior art. This invention identified the expression pattern of gene NU3 in rice and obtained a loss-of-function mutant using gene editing technology. The results showed that the loss-of-function mutant exhibited reduced seedling growth, a significant decrease in thousand-grain weight, reduced seed protein content, and decreased nitrogen utilization rate.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] Technical Solution 1: Application of rice nitrogen utilization gene NU3 or related biological materials in regulating rice growth, wherein regulating rice growth refers to reducing rice nitrogen utilization rate, rice seed quality, and rice seed protein content by silencing rice nitrogen utilization gene NU3; or increasing rice nitrogen utilization rate, rice seed quality, and rice seed protein content by overexpressing rice nitrogen utilization gene NU3.

[0008] The nucleotide sequence of the rice nitrogen utilization gene NU3 is shown in SEQ ID NO.1:

[0009]

[0010] Furthermore, the rice seed quality includes the weight of 1,000 rice seeds.

[0011] The amino acid sequence of the protein encoded by the rice nitrogen utilization gene NU3 is shown in SEQ ID NO.2:

[0012] *

[0013] Furthermore, the relevant biological material is any one of the following A1)-A6):

[0014] A1) contains an expression cassette containing the rice nitrogen utilization gene NU3;

[0015] A2) A recombinant vector containing the rice nitrogen utilization gene NU3;

[0016] A3) Recombinant microorganisms containing the rice nitrogen utilization gene NU3;

[0017] A4) The mutant gene of the rice nitrogen utilization gene NU3;

[0018] A5) Targeting the rice nitrogen utilization gene NU3 with a CRISPR / Cas9-gRNA expression plasmid;

[0019] A6) A recombinant microorganism containing the CRISPR / Cas9-gRNA expression plasmid described in A5).

[0020] Furthermore, the method for regulating rice growth includes:

[0021] By knocking out the rice nitrogen utilization gene NU3, or by introducing the relevant biological materials described in any one of A4)-A6) into rice plants, the nitrogen utilization rate of rice, the seed quality and protein content of rice can be reduced.

[0022] Alternatively, the related biological material described in any one of the rice nitrogen utilization genes NU3 or A1-A3 can be introduced into rice plants to improve the nitrogen utilization rate of rice, improve rice seed quality and protein content.

[0023] Technical Solution 2: A method for regulating rice growth, comprising the steps of knocking out the rice nitrogen utilization gene NU3, thereby reducing the expression level of the NU3 gene in the rice, and thus reducing the rice nitrogen utilization rate, rice seed quality and protein content;

[0024] The nucleotide sequence of the rice nitrogen utilization gene NU3 is shown in SEQ ID NO.1.

[0025] Technical Solution 3: A mutant gene of rice nitrogen utilization gene NU3, wherein the mutant gene is nu3-1 or nu3-2;

[0026] The nucleotide sequence of nu3-1 is shown in SEQ ID NO.12; the nucleotide sequence of nu3-2 is shown in SEQ ID NO.13;

[0027] The amino acid sequence of the protein encoded by nu3-1 is shown in SEQ ID NO.14; the amino acid sequence of the protein encoded by nu3-2 is shown in SEQ ID NO.15.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] This invention identified a rice nitrogen utilization gene, NU3. Knockout of this gene resulted in reduced seedling growth, significantly decreased thousand-grain weight, lower seed protein content, and decreased nitrogen use efficiency in the mutant. Therefore, the NU3 gene plays a crucial role in regulating rice growth, including nitrogen use efficiency, yield, and rice quality. This invention provides new genetic resources and a research foundation for rice nitrogen metabolism research. Attached Figure Description

[0030] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0031] Figure 1 A diagram illustrating the spatiotemporal expression patterns of the NU3 gene in different tissues of rice.

[0032] Figure 2 The expression pattern of the NU3 gene in response to nitrogen (N) element;

[0033] Figure 3 Map of CRISPR-Cas9 knockout vectors containing the NU3 gene target site sequence;

[0034] Figure 4 This is a schematic diagram of gene editing of NU3 in the Nipponbare (NIP) background.

[0035] Figure 5 Comparison of plant morphology of hydroponic seedlings of wild-type NIP and mutants nu3-1 and nu3-2 under low nitrogen (A) and high nitrogen (B) treatments;

[0036] Figure 6 Figure 1 shows the seedling height (A), fresh weight (B), and dry weight (C) of wild-type NIP seedlings and mutants nu3-1 and nu3-2 under different nitrogen treatments.

[0037] Figure 7 The graph shows the results of detecting the thousand-grain weight (A) and protein content (B) of wild-type NIP mutants nu3-1 and nu3-2 under different nitrogen treatments. Detailed Implementation

[0038] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0039] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0040] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0041] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0042] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0043] The nucleotide sequence of the rice nitrogen utilization gene NU3 in this invention is shown in SEQ ID NO.1:

[0044]

[0045] The amino acid sequence of the protein encoded by the rice nitrogen utilization gene NU3 is shown in SEQ ID NO.2:

[0046] *

[0047] Example 1: Exploration of the expression pattern of the rice nitrogen utilization gene NU3

[0048] To obtain the expression pattern of the rice nitrogen utilization gene NU3, RNA was extracted from different tissues (roots, stems, leaves, and panicles) and seeds at different developmental stages (5, 10, 15, 20, 25, and 30 DAF), with three replicates for each sample. RNA extraction from rice seeds followed the same method as for maize endosperm RNA extraction, using the SDS-TRIZOL method (Joao Leiva, Ricardo Dante, and David Holding). RNA extraction from other rice tissues was performed using the TRIZOL method.

[0049] The specific steps are as follows:

[0050] (1) Reagent preparation and sample preparation

[0051] The SDS-buffer (50mM TRIS pH 8.0, 150mM LiCl, 5mM EDTA pH 8.0, 1% SDS) and the containers and pipette tips used were all made with DEPC water. The corresponding samples were taken from the -80℃ freezer and placed on ice for later use. The glumes were quickly removed with tweezers and then placed into 1.5mL RNA-free centrifuge tubes placed on ice. 3-5 seeds were taken from each sample period.

[0052] (2) SDS-buffer extraction

[0053] Add 400 μL of SDS-buffer to the centrifuge tubes. Prepare a steel ball heated over an alcohol flame and add one ball to each centrifuge tube. Immediately place the tubes into a DNA extraction sample shaker and crush the sample quickly for 1 minute. Add 800 μL of phenol (equilibrated at TRIS pH 8.0)-chloroform (1:1), shake well, place on ice for 5 minutes, and centrifuge at 10,000g, 4°C for 10 minutes. Transfer 500 μL of the supernatant to a new RNA-free 1.5 mL centrifuge tube.

[0054] (3) TRIZOL extraction

[0055] Add 1 mL of TRIZOL extract to the sample; add 200 μL of chloroform, shake well, and incubate at room temperature for 3 min; centrifuge at 10,000 g and 4 °C for 10 min.

[0056] (4) RNA precipitation

[0057] Carefully aspirate 500 μL of supernatant into a new RNA-free 1.5 mL centrifuge tube, add 400 μL of isopropanol, mix by inverting, and place on ice for 10 min; centrifuge at 10,000 g, 4 °C for 10 min.

[0058] (5) RNA washing and dissolution

[0059] Carefully discard the supernatant, add 500 μL of 75% ethanol (prepared with DEPC water), and gently suspend the RNA precipitate at the bottom; centrifuge at 10,000g, 4℃ for 5 min, carefully discard the supernatant, centrifuge briefly again for 15 s, use an RNA-free pipette tip to remove the remaining liquid, and then place it in a clean bench to air dry; add 40 μL of DEPC water, and gently tap to promote the dissolution of the RNA precipitate.

[0060] (6) RNA digestion (removal of gDNA contamination)

[0061] The reaction system consisted of 40 μL of template RNA, 4.5 μL of 10× Buffer, and 4 μL of DNase I (RNase-Free). The reaction mixture was treated in a metal bath at 37°C for 30 min and then at 75°C for 5 min. The mixture was then stored at -80°C.

[0062] (7) Synthesis of first-strand cDNA

[0063] cDNA was obtained by reverse transcription using the ReverTraAce qPCR RT Kit (Toyobo, Osaka, Japan). An equal volume of digested RNA was added to each RNase-free centrifuge tube, followed by RNase-free water. The reverse transcription mixture was as follows: Total RNA (2 μg), RNA-free water added to 22 μL. Reaction conditions: 75°C in a metal bath for 5 minutes, then immediately placed on ice. Next, 8 μL of 5×Quantiscript RTBuffer, 4 μL of dNTPs, 2 μL of Oligdt(20), 2 μL of RNA inhibitor, and 2 μL of ReverTraAce were added to each centrifuge tube, mixing to a total volume of 40 μL. The reaction was terminated by incubation at 42°C for 60 minutes, followed by incubation at 75°C for 5 minutes. After the reaction, an equal volume of RNA-free water was added to dilute the mixture by half, and the cDNA template was obtained and stored at -20°C for later use.

[0064] (8) Real-time quantitative PCR

[0065] The kit used was SYBR Green Real-time PCR Master Mix (Toyobo). The reaction mixture consisted of 10 μL of 2×SYBR Premix Ex Taq II, 2 μL of 10 μM PCR Forward Primer, 2 μL of 10 μM PCR Reverse Primer, 4 μL of cDNA template, and water to a final volume of 20 μL. The PCR program was as follows: 95℃ for 30 s, 95℃ for 5 s, 60℃ for 30 s, for a total of 40 cycles; followed by 95℃ for 15 s, 60℃ for 1 min, and 95℃ for 15 s.

[0066] The PCRForwardPrimer consists of qRT-NU3 F and qRT-NU3 R.

[0067] qRT-NU3 F: ACCTCACTCACCTACATCGC (SEQ ID NO.3);

[0068] qRT-NU3 R:AGCTCTTGTTGCATGCTCTG (SEQ ID NO. 4).

[0069] (9) Results Analysis

[0070] The results of the real-time PCR experiment are based on 2 -△△CT The method was used for calculation and analysis, with the rice Actin1 (Os03g0718150) gene as an internal control. The primer set for the internal control gene was: Actin-F: CCCTCCTGAAAGGAAGTACAGTGT (SEQ ID NO.5); Actin-R: GTCCGAAGAATTAGAAGCATTTCC (SEQ ID NO.6).

[0071] The results are as follows Figure 1 As shown in the figure, the NU3 gene is highly expressed in the ear.

[0072] Example 2: Prediction of expression pattern of NU3 gene in rice roots in response to nitrogen treatment

[0073] Using the RAP-DB website (https: / / rapdb.dna.affrc.go.jp), the RAP_Locus number Os10g0390600 for NU3 was entered. Under the "Expression (RiceXPro)" project, "RXP_5002: Root gene expression profile in response to nutrients" was selected to analyze the expression pattern of the NU3 gene in rice roots in response to nutrients. Then, data on nitrogen (N), phosphorus (P), and potassium (K) were downloaded for analysis. The results showed that the expression level (Cy3 signal intensity) of NU3 in rice roots significantly increased under the "-N" 6-hour and 24-hour conditions. Figure 2 This indicates that NU3 is related to nitrogen absorption and utilization.

[0074] Example 3: Construction of a rice nitrogen utilization gene NU3 knockout vector

[0075] 1. Selection of gRNA target sequences

[0076] According to the CRISPR / Cas9 experimental method, the 5′-GGAGGAGAAGTAGGTAGG-3′ sequence containing NGG as the recognition site was selected as the knockout target on the second exon of the NU3 gene (SEQ ID NO.7), and the PAM sequence is AGG.

[0077] 2. Design of upstream and downstream primers for gRNA oligonucleotide chains

[0078] The upstream primer was NU3_F: 5′-TGTGTGGGAGGAGAAGTAGGTAGG-3′ (SEQ ID NO.8);

[0079] The downstream primer was NU3_R: 5′-ACCCTCCTCTTCATCCATCCCAAA-3′ (SEQ ID NO.9).

[0080] 3. Construction of CRISPR / Cas9 vectors

[0081] This embodiment uses a plant Cas9 / gRNA vector construction kit (Catalog. No. BGK032) to load the target sequence and form a recombinant vector containing the NU3 gene target. Figure 3 The specific operating method is as follows:

[0082] (1) Formation of oligo dimers

[0083] Take 5 μL of each of the 10 μM target upstream and downstream primers and add 15 μL of ddH2O. After mixing, treat at 95 °C for 3 min, slowly cool from 95 °C to 25 °C, and treat at 16 °C for 5 min to obtain the double-stranded sequence (oligo dimer) containing the knockout target site.

[0084] (2) The oligo dimer was inserted into the vector.

[0085] Take 1 μL of Cas9 / gRNA vector, 1 μL of oligo dimer from step (1), 1 μL of buffer I and 1 μL of buffer II, add 6 μL of ddH2O, and react in a metal bath at 16℃ for 2 hours.

[0086] (3) Escherichia coli transformation

[0087] Take 10 μL of the final product from step (2) and add it to 50 μL of freshly thawed DH5α competent cells. Gently mix, incubate on ice for 30 min, heat shock at 42°C for 45 s, and let stand on ice for 2 min. Then add 200 μL of antibiotic-free LB and place in a constant temperature shaker at 37°C at 200 rpm. After one hour of recovery, spread it on a natriuretic (Kana+) resistant plate.

[0088] (4) Bacterial PCR detection

[0089] The following day, single colonies were picked and inoculated into kanamycin-resistant liquid medium and cultured in a shaker at 37°C until the bacterial culture became turbid. Sequencing was performed using the BGK032 vector-specific sequencing primers provided in the kit. The sequencing results were analyzed using Snapgene software, and plasmids of positive clones were extracted for later use.

[0090] 4. Agrobacterium-mediated transformation and genetic transformation of rice

[0091] The successfully constructed plasmid was transformed into Agrobacterium (EHA105): 1 μL of plasmid was injected into competent Agrobacterium cells that had been frozen and thawed on ice, then placed on ice for 5 min, in liquid nitrogen for 5 min, at 37℃ for 5 min, and 300 μL of antibiotic-free LB was added and the cells were thawed at 28℃ for 4 h. The plasmid was then evenly spread on a (kanamycin + rifampin) K+ / Rif resistant plate and cultured at 28℃ for 3 days. Single colonies were picked, and positive clones were obtained by detection with hygromycin primers. The positive clones were then expanded in 3 mL of liquid K+ / Rif medium. The positive K+ / Rif bacterial culture was sent to Wuhan Aidijing Biotechnology Co., Ltd. for genetic transformation of rice under the Nipponbare background to obtain rice NU3 gene knockout lines.

[0092] Example 4: Identification of homozygous knockout lines of the rice NU3 gene

[0093] To identify the rice NU3 gene knockout lines obtained in Example 2, the transgenic seedlings were cultured in a room temperature and light incubator for one week, and then positive seedlings were identified. The specific steps are as follows:

[0094] 1. Detection of knockout genetically modified seedlings

[0095] Twenty T0 generation transgenic seedlings were obtained and cultured in a room temperature and light incubator for one week. DNA was extracted from the 20 seedlings, amplified by PCR using NU3TF and NU3TR, and then sequenced. Analysis of the sequencing results revealed two transgenic plants with premature termination of protein translation: NU3-1 and NU3-2, two homozygous mutants with deletion and insertion of the NU3 gene obtained by knockout. The nucleotide sequences of the NU3 gene in the two mutant plants are shown in SEQ ID NO.12 and SEQ ID NO.13, respectively, and the amino acid sequences of the proteins they encode are shown in SEQ ID NO.14 and SEQ ID NO.15, respectively.

[0096] NU3TF: 5′-AGATGAGAGGAGATGAGACG-3′ (SEQ ID NO. 10);

[0097] NU3TR: 5′-GCAGCGGAAGAAGATCAGTG-3′ (SEQ ID NO. 11);

[0098] SEQ ID NO.12(nu3-1):

[0099]

[0100] SEQ ID NO.13(nu3-2):

[0101]

[0102] SEQ ID NO.14(nu3-1):

[0103] MRGDETSSLLFIIHGRLA*;

[0104] SEQ ID NO.15(nu3-2):

[0105] MRGDETSSLLFIPMAA*.

[0106] After obtaining stable T2 knockout lines, further sequencing of the target site yielded homozygous mutants NU3-1 and NU3-2. Figure 4 They harvested mature rice seeds.

[0107] Example 5: Identification of nitrogen use capacity in rice NU3 gene knockout seedlings

[0108] Wild-type (NIP) and mutant (nu3-1, nu3-2) plants were subjected to hydroponic experiments with different nitrogen gradients to detect the growth characteristics of the plants under different nitrogen levels.

[0109] 1. The hydroponic experiment method is as follows:

[0110] (1) Wash the seeds with 75% anhydrous ethanol for 1 min, then soak the seeds in 50% sodium hypochlorite for 30 min, then wash them thoroughly with distilled water, and then soak them in distilled water at 37°C for 24 hours.

[0111] (2) After soaking, wrap the seeds with a damp towel and place them at 37°C in the dark for 48 hours to germinate.

[0112] (3) The standard nitrogen nutrient solution formula was formulated according to the recommendations of the International Rice Research Institute (IRRI). Two nitrogen concentrations were set in this experiment: low nitrogen (no nitrogen) and high nitrogen (4 times the recommended standard nitrogen content). The contents of other elements were formulated according to the recommended standards. The standard nitrogen nutrient solution formula is shown in Table 1.

[0113] (4) After germination, select seeds with similar sprout and root lengths and sow them in a hydroponic box. Cultivate them in a 25℃ light incubator (12h light, 12h darkness).

[0114] (5) The hydroponic method is as follows: 3 days of hydroponics, 4 days of standard nitrogen nutrient solution culture, and 7 days of nutrient solution culture with different nitrogen concentrations, for a total of 14 days. The nutrient solution is changed every 3 days.

[0115] Precautions for hydroponics:

[0116] 1) The water temperature should not be too high or too low, generally around 25℃;

[0117] 2) The roots should be kept away from light to prevent algae growth and root respiration losses;

[0118] 3) When culturing in large bodies of water, the pH of the culture medium should be between 5 and 6, and should not be lower than 4.

[0119] 4) Large-volume culture requires water circulation to avoid the precipitation of trace elements such as iron under low oxygen conditions;

[0120] 5) For large-volume culture, the culture medium can be changed every 3-4 weeks, while for small-volume culture, it should be changed every 3-5 days. If the seedlings turn yellow, nitrogen should be added in time.

[0121] (6) After 14 days of hydroponics, 10 representative plants with basically the same growth were randomly selected to examine the seedling height, fresh weight and dry weight, and the results were repeated three times.

[0122] Table 1. Hydroponic Nutrient Solution Formula (Recommended Standard Nitrogen Content)

[0123]

[0124] Note: All of the above are stock solutions. They need to be diluted 1000 times when preparing the working solution. The amount of macro-elements used during the seedling stage is 1 / 4 of the normal amount. For micro-element stock solutions, add 500mL of concentrated sulfuric acid and 10-15g of EDTA per 10L. The pH needs to be adjusted to around 5.5 when preparing the working solution.

[0125] 2. Results of the hydroponic experiment:

[0126] 2.1 Comparison of plant architecture between wild-type (NIP) and mutant plants under nitrogen-free (N0) and 4-fold nitrogen (N4) treatments. Figure 5 As shown.

[0127] 2.2 Results of agronomic traits of wild-type (NIP) and mutant plants under nitrogen-free (N0) and 4-fold nitrogen (N4) treatments are as follows: Figure 6 As shown in the figure, under low nitrogen conditions, the mutant plants showed no significant difference in plant height compared to the wild-type NIP. Under high nitrogen conditions, the mutant nu3-1 showed no significant difference in plant height compared to the wild-type, while the nu3-2 plant height was significantly lower than that of the wild-type. Figure 6 (A) Under low-nitrogen and high-nitrogen conditions, the fresh weight and dry weight of both mutants were significantly lower than those of the wild type. Figure 6 B and Figure 6 (C in the text). The results indicate that the NU3 gene mutation may have altered the nitrogen use efficiency of rice, leading to a decrease in growth.

[0128] Example 6: Determination of 1000-grain weight and protein content of rice seeds

[0129] 1. Thousand-grain weight determination

[0130] Stable and homogeneous T2 generation mutant plants and wild-type (NIP) plants were sown in low-nitrogen (LN) and high-nitrogen (HN) field environments. The low-nitrogen field was treated with 75 kg / hm² of urea (containing 46% N), and the high-nitrogen field with 180 kg / hm² of urea. Phosphorus and potassium fertilizers were applied in equal amounts. Mature rice seeds were harvested. The mature seeds were dried in a 65℃ oven to constant weight. Seed morphology analysis was performed: 100 randomly selected mature and plump seeds were weighed, with three replicates per sample. The average weight was multiplied by 10 to obtain the final thousand-seed weight. Wild-type seeds were used as a control. The analysis results are as follows: Figure 7 As shown in B, it can be seen that the thousand-grain weight of the mutant was significantly lower than that of the wild type under both low-nitrogen and high-nitrogen conditions.

[0131] 2. Determination of total protein content in seeds

[0132] First, the nitrogen content of mature seeds of wild type and mutant was determined using a SEALAutoAnalyzer 3 rheometer. Then, the protein content was calculated based on the nitrogen content data using the following formula:

[0133] Protein content (%) = C0 × 0.1 × 5.95 ÷ (200 × (1 - water content)) × 100

[0134] C0 represents the nitrogen content (mg / L) measured by a rheometer; the moisture content of rice noodles is generally around 8%, and this example is calculated based on 8%.

[0135] The method for determining the nitrogen content of seeds is as follows:

[0136] (1) Weigh 200 mg of the sample to be tested and place it in a digestive tube. Each sample is repeated 3 times.

[0137] (2) Add 5 mL of H2SO4 to the digestion tube, digest at 290℃ for 20 min, and remove the digestion tube after boiling and shake well.

[0138] (3) Continue digestion for 1 hour, shaking once every 20 minutes;

[0139] (4) Remove the digestion tube and let it cool to room temperature. Add 2 mL of H2O2 and shake well.

[0140] (5) Digest again for 10 minutes. After the digestion is complete, observe whether the sample solution becomes clear. If it becomes clear, remove the digestion tube and cool it to room temperature. If the sample still has color, cool it to room temperature and add 200 μL of H2O2. Shake well and digest again for 10 minutes. Repeat this step until the sample solution becomes clear.

[0141] (6) Add ddH2O to the digestion tube cooled to room temperature and bring the volume up to 100 mL;

[0142] (7) Before testing, shake the sample well, let it stand for 20 minutes, then use a sample cup to collect the supernatant of the sample and measure it using a SEALAutoAnalyzer 3 rheometer. Calculate the nitrogen content using the following formula:

[0143] Nitrogen content (mg / g) = C0 × 0.1 ÷ 0.2

[0144] Where C0 is the N content (mg / L) measured by the rheometer; 0.1 is the volume of 100 mL of the sample to be tested, which is converted to 0.1 L; and 0.2 is the sample mass.

[0145] Protein content test results as follows Figure 7 As shown in A, it can be seen that the protein content in the mutant seeds was significantly lower than that in the wild type under both low-nitrogen and high-nitrogen conditions.

[0146] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. Rice nitrogen utilization genes NU3 The application of its related biomaterials in regulating rice growth, characterized in that, The regulation of rice growth refers to silencing the nitrogen utilization gene in rice. NU3 This reduces the protein content of rice seeds; The rice nitrogen utilization gene NU3 The nucleotide sequence is shown in SEQ ID NO.1; The relevant biological material is any one of A1) or A2) below: A1) Targeting the rice nitrogen utilization gene NU3 CRISPR / Cas9-gRNA expression plasmid; A2) Recombinant microorganisms containing the CRISPR / Cas9-gRNA expression plasmid described in A1).

2. The application according to claim 1, characterized in that, The rice nitrogen utilization gene NU3 The amino acid sequence encoding the protein is shown in SEQ ID NO.

2.

3. The application according to claim 1, characterized in that, The methods for regulating rice growth include: By introducing the relevant biological material as described in claim 1(A1) into rice plants, the protein content of rice is reduced.

4. A method for regulating rice growth, characterized in that, Including knocking out nitrogen utilization genes in rice NU3 To make the rice NU3 The process of reducing gene expression levels, thereby reducing the protein content of rice seeds; The rice nitrogen utilization gene NU3 The amino acid sequence encoding the protein is shown in SEQ ID NO.2.

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