Application of Rice Nitrogen Utilization Gene NU4 in Regulating Rice Growth

Abstract

The present invention discloses the application of the rice nitrogen utilization gene NU4 in regulating rice growth, belonging to the technical field of plant genetic engineering. The nucleotide sequence of the rice nitrogen utilization gene NU4 of the present invention is shown in SEQ ID NO.1; the regulation of rice growth refers to regulating the nitrogen utilization rate of rice, regulating the seed quality of rice, and regulating the yield of rice. The present invention identified the expression pattern of the nitrogen metabolism gene NU4 in rice and obtained its loss-of-function mutant using gene editing technology. The seeds of this loss-of-function mutant have a narrower grain thickness, resulting in a significant decrease in 1000-grain weight; the seed protein content decreases; at the same time, the nitrogen content of the mutant rice seedlings increases, resulting in a decrease in the nitrogen utilization rate of rice.

Description

Technical Field

[0001] The present invention relates to the technical field of plant genetic engineering, and particularly to the application of rice nitrogen utilization gene NU4 in regulating rice growth. Background Art

[0002] As one of the main food crops, the yield of rice has always been concerned by all sectors of society. Nitrogen, as one of the three major mineral elements of crops, has a significant impact on the yield of rice. The widespread use of nitrogen fertilizers has increased the yields of many cereals, while the overuse of nitrogen fertilizers will cause serious environmental pollution problems, such as soil acidification and water eutrophication. Therefore, in order to increase food production and alleviate the environmental problems caused by the overuse of nitrogen fertilizers, improving the nitrogen use efficiency of rice has become a practical approach.

[0003] According to research, as one of the main fertilizers in agricultural production, only 30%-50% of nitrogen fertilizer can be absorbed by crops (Raun et al., Improving Nitrogen Use Efficiency for Cereal Production. Agronomy Journal, 1999, 91.). Therefore, in order to meet the nitrogen fertilizer demand of crops and reduce the environmental pollution problems caused by excessive use of nitrogen fertilizer, it is very important to increase the nitrogen use efficiency of plants. A large number of genes related to nitrogen utilization have been reported. For example, NRT1.1B promotes efficient nitrogen utilization by triggering nitrate response (Hu et al., Nitrate–NRT1.1B–SPX4 cascade integrates nitrogen and phosphorus signalling networks in plants. Nat. Plants, 2019, 401–413.); OsNR2 has high NR enzyme activity, promotes hypochlorite sensitivity and nitrate absorption, thereby increasing nitrogen use efficiency and promoting rice yield improvement (Gao et al., The Indica nitrate reductase gene OsNR2 allele enhances rice yield potential and nitrogen use efficiency. Nat Commun, 2019, 10, 5207.); OsAlaAT1 regulates the effective utilization of nitrogen by regulating the carbon-nitrogen metabolic balance of rice, improving rice yield and quality (Fang et al., Alanine aminotransferase (OsAlaAT1) modulates nitrogen utilization, grain yield, and quality in rice. Journal of Genetics and Genomics, 2022, 49:510-513.). However, there are still a large number of unknowns about the genes related to nitrogen utilization in rice and their applications, and further exploration is needed to increase nitrogen use efficiency and provide excellent gene resources for high nitrogen metabolism in rice nitrogen metabolism. Summary of the Invention

[0004] The object of the present invention is to provide the application of rice nitrogen utilization gene NU4 in regulating rice growth, so as to solve the problems existing in the above-mentioned prior art. The present invention identified the expression pattern of nitrogen metabolism gene NU4 in rice and obtained its loss-of-function mutants by using gene editing technology. The seeds of the loss-of-function mutants have a narrower grain thickness, resulting in a significant decrease in 1000-grain weight; the protein content of the seeds decreases; at the same time, the nitrogen content of the mutant rice seedlings increases, resulting in a decrease in the nitrogen utilization rate of rice.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides the application of rice nitrogen utilization gene NU4 or biological materials related to NU4 in regulating rice growth, and the regulation of rice growth refers to regulating the nitrogen utilization rate of rice, regulating the seed quality of rice, and regulating the yield of rice;

[0007] The nucleotide sequence of the rice nitrogen utilization gene NU4 is shown in SEQ ID NO.1:

[0008]

[0009] Preferably, the amino acid sequence of the protein encoded by the rice nitrogen utilization gene NU4 is as shown in SEQ ID NO.2:

[0010] MSYNHSPSITAETINQKVRIFTYEPCGEIVRHARRLEKEIYENPGSLPFQEIIYCNLGNPQALGQRPINFFREVLSLCDNPSLIDRDEARALFSPCALKRARKIIESLPGRDSGSYTSSQGVRGLREAVADGIAARDGFPSKPDNIFLTDGASSAINMMMQILIRSHEDGILCPLPEYPLYSASIILHGGTMVPYNLTEDSIWGLEIFEVKRCLEDARASGLTIRAMVVINPGNPTGQVLSITNQEEIVEFCRKEGLVILADEVYQENVYTENKRFNSFKKVARSLGYDHHDLSIVSFHSVSMGYYGECGRRGGYMEICGFGDDVIDEMYKLASLTICPNIAGQILISLVMDPPKLGDEAFEIFMVEKEETYSSLLKRAKALQKAFNGLEGVSCNKFEGAMYLFPRLRLPQAAIKAAQLEGVSPDVFYAHRLLDATGIAVVPGSGFHPVSGTSHIRCTILPGEETITAMVPSLQAFHEAFMDEFRG*。

[0011] Preferably, the biological material related to NU4 is any one of the following B1)-B6):

[0012] B1) An expression cassette containing the rice nitrogen utilization gene NU4;

[0013] B2) A recombinant vector containing the rice nitrogen utilization gene NU4;

[0014] B3) A recombinant microorganism containing the rice nitrogen utilization gene NU4;

[0015] B4) A mutant gene of the rice nitrogen utilization gene NU4;

[0016] B5) A CRISPR / Cas9-gRNA expression plasmid targeting the rice nitrogen utilization gene NU4;

[0017] B6) A recombinant microorganism containing the expression plasmid described in B5).

[0018] Preferably, the method for regulating rice growth is:

[0019] By knocking out the rice nitrogen utilization gene NU4, or introducing the biological material related to NU4 as described in any one of B4)-B6) into rice plants, the nitrogen utilization rate of rice, the quality of rice seeds or the yield of rice is reduced;

[0020] Or, introducing the rice nitrogen utilization gene NU4 or the biological material related to NU4 as described in any one of B1)-B3) into rice plants to improve the nitrogen utilization rate of rice, improve the quality of rice seeds or increase the yield of rice.

[0021] The present invention also provides a mutant gene of the rice nitrogen utilization gene NU4, and the mutant gene is nu4-1 or nu4-2;

[0022] The nucleotide sequence of nu4-1 is shown in SEQ ID NO.12; the nucleotide sequence of nu4-2 is shown in SEQ ID NO.13;

[0023] The amino acid sequence of the protein encoded by nu4-1 is shown in SEQ ID NO.14; the amino acid sequence of the protein encoded by nu4-2 is shown in SEQ ID NO.15.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] The present invention has identified a rice nitrogen utilization gene NU4. After knocking out this gene, the mutant seeds show a narrower grain thickness, resulting in a significant decrease in 1000-grain weight; the protein content of the seeds decreases; the nitrogen content of rice seedlings increases, the root length becomes longer, and the enzyme activity becomes higher, resulting in a decrease in the nitrogen utilization rate of rice. Therefore, the NU4 gene plays a key role in regulating rice growth, including the nitrogen utilization rate of rice, rice yield, rice quality, etc. The present invention provides a new gene resource and research basis for rice nitrogen metabolism research. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required to be used in the embodiments. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0027] Figure 1 It is an analysis diagram of the spatio-temporal expression pattern of the NU4 gene in different tissues of rice;

[0028] Figure 2 It is a schematic diagram of the sequencing results after editing the NU4 gene in the background of Nipponbare (NIP);

[0029] Figure 3 Comparison chart of grain shape analysis results between wild-type NIP and mutants nu4-1 and nu4-2;

[0030] Figure 4 Appearance comparison chart of polished rice between wild-type NIP and mutants nu4-1 and nu4-2;

[0031] Figure 5 Detection result chart of protein content in mature seeds of wild-type NIP and mutants nu4-1 and nu4-2;

[0032] Figure 6 Plant type comparison chart of hydroponic seedlings of wild-type NIP and mutants nu4-1 and nu4-2 under different nitrogen treatments;

[0033] Figure 7 Detection result chart of agronomic traits of wild-type NIP and mutants nu4-1 and nu4-2 under different nitrogen treatments;

[0034] Figure 8 Detection result chart of nitrogen content in seedlings of wild-type NIP and mutants nu4-1 and nu4-2 under different nitrogen treatments;

[0035] Figure 9 Detection result chart of α-ketoglutaric acid (α-KG) content in roots of hydroponic seedlings of wild-type NIP and mutants nu4-1 and nu4-2;

[0036] Figure 10 Detection result chart of glutamate synthase (GOGAT) enzyme activity in roots of hydroponic seedlings of wild-type NIP and mutants nu4-1 and nu4-2. Detailed implementation manners

[0037] Now, various exemplary implementation manners of the present invention will 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, characteristics, and implementation schemes of the present invention.

[0038] It should be understood that the terms described in the present invention are only for describing specific implementation manners and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0039] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although this invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of this invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the said documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0040] Without departing from the scope or spirit of the present invention, various modifications and variations can be made to the specific embodiments of the description of the present invention, which will be obvious to those skilled in the art. Other embodiments obtained from the description of the present invention will be obvious to those skilled in the art. The description and examples of the present invention are merely exemplary.

[0041] Regarding the use of "comprising", "including", "having", "containing", etc. herein, they are all open-ended terms, meaning including but not limited to.

[0042] Example 1 Exploration of the Expression Pattern of the Rice Nitrogen Utilization Gene NU4

[0043] To obtain the expression pattern of the rice nitrogen utilization gene NU4, RNA was extracted from different tissue parts of rice (roots, stems, leaves, panicles) and seeds at different developmental stages (5, 10, 15, 20, 25, 30 DAF), and three replicates were set for each sample. Among them, the extraction of rice seed RNA referred to the method for extracting maize endosperm RNA, using the SDS-TRIZOL method (Joao Leiva, Ricardo Dante and David Holding), and the extraction of RNA from other tissues of rice all used the TRIZOL method.

[0044] The specific steps are as follows:

[0045] (1) Reagent Preparation and Sample Preparation

[0046] Prepare SDS-buffer (50 mM TRIS pH 8.0, 150 mM LiCl, 5 mM EDTA pH 8.0, 1% SDS), and the containers and pipette tips used are all treated with DEPC water; the corresponding samples taken out from the -80 °C refrigerator are placed on ice for use. Use tweezers to quickly remove the glumes, and then put them into 1.5 mL RNA-free centrifuge tubes placed on ice. For each period, 3 - 5 seeds of the sample are sufficient.

[0047] (2) SDS-buffer Extraction

[0048] Add 400 μL of SDS-buffer to the above centrifuge tubes. Prepare steel beads that have been heated on an alcohol flame, add one bead to each centrifuge tube, immediately place them in a DNA extraction sample shaker, and quickly crush the samples for 1 min. Add 800 μL of phenol (equilibrated in TRIS pH 8.0)-chloroform (1:1), shake well, place on ice for 5 min, and centrifuge at 10,000 g at 4 °C for 10 min. Pipette 500 μL of the supernatant into a new 1.5 mL RNA-Free centrifuge tube.

[0049] (3) TRIZOL Extraction

[0050] Add 1 mL of TRIZOL extraction solution to the sample; add 200 μL of chloroform, shake well, and let stand at room temperature for 3 min; centrifuge at 10,000 g at 4 °C for 10 min.

[0051] (4) RNA Precipitation

[0052] Carefully pipette 500 μL of the supernatant into a new 1.5 mL RNA-free centrifuge tube, add 400 μL of isopropanol, mix by inverting up and down, and place on ice for 10 min; centrifuge at 10,000 g at 4 °C for 10 min.

[0053] (5) RNA Washing and Dissolution

[0054] Carefully pour off the supernatant, add 500 μL of 75% ethanol (prepared with DEPC water), gently suspend the RNA precipitate at the bottom; centrifuge at 10,000 g at 4 °C for 5 min, carefully pour off the supernatant, centrifuge briefly for 15 s, use an RNA-free pipette tip to aspirate the residual liquid, and then air-dry in a laminar flow hood; add 40 μL of DEPC water, flick gently to promote the dissolution of the RNA precipitate.

[0055] (6) RNA Digestion (Removing gDNA Contamination)

[0056] The reaction system is 40 μL of Template RNA, 4.5 μL of 10× Buffer, and 4 μL of DNase I (RNase-Free). Treat with a metal bath at 37 °C for 30 min, 75 °C for 5 min, and store in a -80 °C refrigerator.

[0057] (7) First Strand cDNA Synthesis

[0058] Reverse transcription was performed using the ReverTraAce qPCR RT Kit (Toyobo, Osaka, Japan) to obtain cDNA. Equal amounts of digested RNA were added to each RNase-free centrifuge tube, and RNase-free water was added and mixed well. The reverse transcription system was as follows: Total RNA (2 μg), RNA-free water was added to make up 22 μL. Reaction conditions: Treated with a metal bath at 75 °C for 5 minutes, and immediately placed on ice for later use; Then, 8 μL of 5×Quantiscript RT Buffer, 4 μL of dNTP, 2 μL of Oligdt(20), 2 μL of RNA inhibitor, and 2 μL of ReverTraAce were added to each centrifuge tube to make a reaction system with a total volume of 40 μL. The reaction conditions were incubation at 42 °C for 60 min and incubation at 75 °C for 5 min to terminate the reaction. After the reaction was completed, an equal volume of RNA-free water was added to dilute it by one-fold, and after mixing, the cDNA template was obtained and stored at -20 °C in the refrigerator for later use.

[0059] (8) Real-time fluorescence quantitative PCR

[0060] The kit used was SYBR Green Real-time PCR Master Mix (Toyobo). The reaction system: 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 was added to make up 20 μL. The PCR program was: 95 °C for 30 s, 95 °C for 5 s, 60 °C for 30 s, with 40 cycles; 95 °C for 15 s, 60 °C for 1 min, 95 °C for 15 s.

[0061] Among them, PCR Forward Primer: qRT-NU4 F: AGCGTTCAACGGTTTGGAAG (SEQ ID NO.3);

[0062] PCR Reverse Primer: qRT-NU4 R: GTGGGAAGAGGTACATGGCT (SEQ ID NO.4).

[0063] (9) Result analysis:

[0064] The results of the Real-time PCR experiment were based on 2 -△△CTCalculated and analyzed by the method, the rice Actin1 (Os03g0718150) gene was used as an internal reference. The primer set for the internal reference gene was: Actin-F: CCCTCCTGAAAGGAAGTACAGTGT (SEQ ID NO.5); Actin-R: GTCCGAAGAATTAGAAGCATTTCC (SEQ ID NO.6).

[0065] The results are as Figure 1 shown. It can be seen that the NU4 gene was highly expressed at the endosperm stage (20 DAF).

[0066] Example 2 Construction of the rice nitrogen utilization gene NU4 knockout vector

[0067] 1. Selection of the gRNA target sequence

[0068] According to the CRISPR / Cas9 experimental method, the sequence 5′-TCACCTACGAGCCATGTGGG-3′ containing NGG as the recognition site on the second exon of the NU4 gene was selected as the knockout target (SEQ ID NO.7), and the PAM sequence was GGG.

[0069] 2. Design of the upstream and downstream primers of the gRNA oligonucleotide chain

[0070] The upstream primer was NU4_F: 5′-TGTGTGTCACCTACGAGCCATGT-3′ (SEQ ID NO.8);

[0071] The downstream primer was NU4_R: 5′-AAACACATGGCTCGTAGGTGACA-3′ (SEQ ID NO.9).

[0072] 3. Construction of the CRISPR / Cas9 vector

[0073] In this example, a plant Cas9 / gRNA vector construction kit (Catalog.No.BGK032) was used to load the target sequence to form a recombinant vector containing the NU4 gene target. The specific operation method was as follows:

[0074] (1) Formation of the oligo dimer

[0075] Take 5 μL of each of the 10 μM upstream and downstream primers of the target 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 a double-stranded sequence (oligo dimer) containing the knockout target site.

[0076] (2) Insertion of the oligo dimer into the vector

[0077] Take 1 μL of the Cas9 / gRNA vector, 1 μL of the oligo dimer in step (1), 1 μL each of buffer one and buffer two, add 6 μL of ddH2O, and react on a metal bath at 16 °C for 2 hours.

[0078] (3) Escherichia coli transformation

[0079] Take 10 μL of the final product in step (2) and add it to 50 μL of freshly thawed DH5a competent cells. Flick gently to mix, incubate on ice for 30 min, heat shock at 42 °C for 45 s, let stand on ice for 2 min, then add 200 μL of antibiotic-free LB, place in a 37 °C constant temperature shaker at 200 rpm, and after one hour of recovery, spread it on a plate with kanamycin resistance (Kana+).

[0080] (4) Bacterial liquid PCR detection

[0081] The next day, pick monoclonal colonies and inoculate them into a liquid medium with kanamycin resistance. Culture in a 37 °C shaker until the bacterial liquid becomes turbid. Use the specific sequencing primer for the BGK032 vector provided in the kit for sequencing; use Snapgene software to analyze the sequencing results, extract the plasmid of the positive clone bacterial liquid, and set aside.

[0082] 4. Agrobacterium transformation and rice genetic transformation

[0083] Transfer the successfully constructed plasmid into Agrobacterium (EHA105): Pipette 1 μL of the plasmid into the competent Agrobacterium that has been frozen and thawed on ice, then place it on ice for 5 min, in liquid nitrogen for 5 min, at 37 °C for 5 min, add 300 μL of antibiotic-free LB and recover at 28 °C for 4 h. Spread evenly on a plate with (kanamycin + rifampicin) K+ / Rif resistance. After culturing at 28 °C for 3 days, pick monoclonal colonies, use the hygromycin primer to detect and obtain positive clones, and expand the culture in 3 mL of liquid K+ / Rif medium. Send the positive K+ / Rif bacterial liquid to Wuhan Aidi Jing Biotechnology Co., Ltd. for rice genetic transformation under the background of Nipponbare to obtain the rice NU4 gene knockout line.

[0084] Example 3 Identification of the rice NU4 gene knockout line

[0085] To identify the rice NU4 gene knockout line obtained in Example 2, place the obtained transgenic seedlings in a normal temperature light incubator for one week, and then conduct the identification of positive seedlings. The specific steps are as follows:

[0086] 1. Detection of knockout transgenic seedlings

[0087] Twenty-five T0 generation transgenic seedlings were obtained. After being cultured in a normal temperature light incubator for one week, the DNA of the 25 seedlings was extracted. After PCR amplification with NU4TF and NU4TR and then sequencing, the sequencing results were analyzed, and two transgenic plants with premature termination of protein translation were obtained, namely two homozygous mutants nu4-1 and nu4-2 with insertions and deletions of the knocked-out NU4 gene. The nucleotide sequences of the NU4 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 encoded proteins are shown in SEQ ID NO.14 and SEQ ID NO.15 respectively.

[0088] NU4TF: 5′-ATCGATTGACACGGGTCCGA-3′ (SEQ ID NO.10);

[0089] NU4TR: 5′-CGGTGTCAAACATTTTGAAACG-3′ (SEQ ID NO.11);

[0090] SEQ ID NO.12 (nu4-1):

[0091] ATGTCGTACAACCATTCGCCGTCCATCACCGCAGAGACCATAAACCAGAAGGTCAGGATCTTCACCTACGAGCCATtGTGGGGAGATTGTCCGCCACGCACGGCGGTTAGAGAAGGAGATATACGAAAATCCAGGCTCTCTCCCTTTTCAAGAGATAATATACTGCAATCTTGGGAACCCTCAGGCTCTTGGCCAACGACCAATCAATTTCTTTCGCGAGGTTCTTTCTCTGTGTGA.

[0092] SEQ ID NO.13 (nu4-2):

[0093] ATGTCGTACAACCATTCGCCGTCCATCACCGCAGAGACCATAAACCAGAAGGTCAG GATCTTCACCTACGAGCCTGTGGGGAGATTGTCCGCCACGCACGGCGGTTAG.

[0094] SEQ ID NO.14 (nu4-1):

[0095] MSYNHSPSITAETINQKVRIFTYEPLWGDCPPRTAVREGDIRKSRLSPFSRDNILQSWEPSGSWPTTNQFLSRGSFSV*。

[0096] SEQ ID NO.15(nu4-2):

[0097] MSYNHSPSITAETINQKVRIFTYEPVGRLSATHGG*。

[0098] 2. Phenotypic identification

[0099] Sow the T1 generation transgenic plants in the natural field environment, eliminate the edge row effect, select plants with normal morphology, and choose 5 - 10 stable lines. After obtaining the stably inherited T2 generation transgenic lines, further sequence the target site. The sequencing results are as Figure 2 shown. After determining the target site, harvest the mature rice seeds.

[0100] Put the mature seeds in an oven at 65 °C and dry them to a constant weight. Conduct grain shape analysis on the mature seeds: Randomly select 100 mature and plump seeds for weighing, with 3 replicates for each sample, and calculate the average value × 10 as the final data of the 1000 - grain weight of the seeds; Randomly select 20 mature and plump seeds, use a vernier caliper to measure their grain length, grain width, and grain thickness (require measuring the values at the widest and thickest positions of the seeds as the grain width and grain thickness of the seeds), and calculate the average value as the final data of the grain length, grain width, and grain thickness of the seeds. Using wild - type seeds as a control, the analysis results are as Figure 3 shown. It can be seen that the grain thickness and 1000 - grain weight of the mutants are significantly lower than those of the wild - type, and there is no obvious difference in grain length and grain width.

[0101] Observe the polished rice of the wild - type and mutants. As Figure 4 shown, it can be seen that compared with the wild - type, the mutants show a partial opaque endosperm phenotype.

[0102] 3. Determination of total seed protein content

[0103] First, use a SEAL AutoAnalyzer 3 rheometer to measure the nitrogen content of the mature seeds of the wild - type and mutants respectively, and then calculate the protein content according to the nitrogen content measurement data using the following formula:

[0104] Protein content (%) = C0 × 0.1 × 5.95 ÷ (200 × (1 - moisture content)) × 100

[0105] C0 is the N content (mg / L) measured by the rheometer; The moisture content of rice flour is generally about 8%, and in this example, it is calculated according to 8%.

[0106] Among them, the method for measuring the nitrogen content of seeds is as follows:

[0107] (1) Weigh 200 mg of the sample to be measured and place it in a digestion tube, with 3 replicates for each sample;

[0108] (2) Add 5 mL of H2SO4 to the digestion tube and digest at 290 °C for 20 min. After boiling, take out the digestion tube and shake well;

[0109] (3) Continue to digest for 1 h, shaking well every 20 min;

[0110] (4) Take out the digestion tube. After it cools to room temperature, add 2 mL of H2O2 and shake well;

[0111] (5) Digest again for 10 min. After completion, observe whether the sample solution becomes clear. If it becomes clear, take out the digestion tube and cool it to room temperature. If the sample to be measured still has color, add 200 μL of H2O2 after cooling to room temperature, shake well, and digest again for 10 min. Repeat this step until the sample solution becomes clear;

[0112] (6) Add ddH2O to the digestion tube cooled to room temperature and make the volume up to 100 mL;

[0113] (7) Shake the sample to be measured well before measuring on the machine. After standing for 20 min, take the supernatant of the sample to be measured with a sampling cup and measure it with a SEAL AutoAnalyzer 3 rheometer. The nitrogen content is calculated according to the following formula for the measured data:

[0114] Nitrogen content (mg / g) = C0 × 0.1 ÷ 0.2

[0115] Among them, C0 is the N content (mg / L) measured by the rheometer; 0.1 is the volume of 100 mL of the sample to be measured for volume fixation, converted to 0.1 L; 0.2 is the sample mass.

[0116] The detection results of protein content are as Figure 5 shown. It can be seen that compared with the wild type, the protein content in the mutant seeds decreased significantly.

[0117] Example 4 Identification of nitrogen utilization ability of rice NU4 gene knockout plants

[0118] Hydroponic experiments were carried out on wild-type (NIP) and mutant (nu4-1, nu4-2) plants with different nitrogen gradients respectively to detect the growth characteristics of plants under different nitrogen levels.

[0119] 1. The hydroponic experiment method is as follows:

[0120] (1) Wash the seeds with 75% absolute ethanol for 1 min, then soak the seeds in 50% sodium hypochlorite for 30 min, and then wash them thoroughly with distilled water. After washing, soak them in distilled water at 37 °C for 24 hours;

[0121] (2) After soaking, wrap the seeds with a moist towel and place them at 37 °C for germination in the dark for 48 h;

[0122] (3) The standard nitrogen nutrient solution formula refers to the recommended standard of the International Rice Research Institute (IRRI). Two nitrogen concentrations were set in this experiment, namely low nitrogen (no nitrogen) and high nitrogen (4 times the recommended standard nitrogen content). The content of other elements refers to the recommended standard. The standard nitrogen nutrient solution formula is shown in Table 1;

[0123] (4) After germination, select seeds with relatively consistent bud length and root length and sow them into the hydroponic box, and culture them in a 25 °C light incubator (12 h light, 12 h dark);

[0124] (5) The hydroponic method is as follows: hydroponics for 3 days, standard nitrogen nutrient solution culture for 4 days, and nutrient solution with different nitrogen concentrations for 7 days, a total of 14 days. The nutrient solution is replaced every 3 days;

[0125] Precautions for hydroponics:

[0126] 1). The water temperature should not be too high or too low, generally about 25 °C;

[0127] 2). The roots should be kept away from light to prevent the growth of algae and the loss of root respiration;

[0128] 3). When culturing in a large water body, the pH of the culture solution is between 5 and 6, and the minimum cannot be lower than 4;

[0129] 4). When culturing in a large water body, water circulation is required to avoid precipitation of trace elements such as iron under low oxygen;

[0130] 5). The culture solution can be replaced every 3 - 4 weeks for large water body culture and every 3 - 5 days for small water body culture. If the seedlings turn yellow, nitrogen should be supplemented in a timely manner.

[0131] (6) After 14 days of hydroponics, randomly select 10 representative plants with basically the same growth vigor to investigate the agronomic traits and nitrogen content of the seedlings.

[0132] Table 1 Formula of hydroponic nutrient solution (recommended standard nitrogen content)

[0133]

[0134] Note: All of the above are mother solutions. They need to be diluted 1000 times when prepared into working solutions. The amount of macroelements used in the seedling stage is 1 / 4 of the normal amount. For every 10L of trace element mother solution, add 500mL of concentrated sulfuric acid and 10-15g of EDTA. When prepared into working solution, the pH needs to be adjusted to around 5.5.

[0135] 2. Hydroponic experiment results:

[0136] 2.1 Comparison of plant types between wild type and mutant plants under different nitrogen treatments Figure 6 shown.

[0137] 2.2 Agronomic trait test results of wild-type and mutant plants under different nitrogen treatments Figure 7 As shown. It can be seen that under low nitrogen and high nitrogen conditions, the wild-type plants were significantly higher in plant height, fresh weight and dry weight than the mutants, and the root length was lower than the mutants. This shows that the NU4 gene mutation changes the agronomic traits of rice plants.

[0138] 2.3 Take 5 seedlings that grow basically the same under different nitrogen levels as a group, and measure the nitrogen content of 3 groups of seedlings. The sample to be tested was sterilized at 108℃ for 1 hour, dried at 80℃ to constant weight, and crushed with a grinder after weighing. The seed nitrogen content determination method in Example 3 was used for detection. The test results are as follows: Figure 8 As shown. It can be seen that under low nitrogen and high nitrogen conditions, the nitrogen content in the wild-type seedlings was lower than that in the mutant seedlings, indicating that the NU4 gene mutation changed the absorption and transfer of nitrogen in rice seedlings and affected the nitrogen utilization rate.

[0139] 3. Determination of α-ketoglutarate content and enzyme activity in the roots of hydroponic seedlings

[0140] 3.1 Determination of α-ketoglutaric acid (α-KG) content

[0141] According to the above hydroponic experimental method, the wild type (NIP) and mutant (nu4-1, nu4-2) plants were cultured in a hydroponic nutrient solution with the recommended standard nitrogen content for 15 days. The α-ketoglutarate content in the roots of wild type and mutant seedlings cultured for 15 days was determined using the Grease α-ketoglutarate content determination kit (microplate method). The method was slightly modified with reference to the determination method provided by the kit. The specific method is as follows:

[0142] (1) Prepare the standard sample stock solution and reagents 1-6 (all reagents are provided by the kit) according to the assay method of the kit;

[0143] (2) Take 10 μL of the standard sample stock solution in a 1.5 mL centrifuge tube, add 990 μL of ddH2O, dilute and place on ice as the standard sample;

[0144] (3) Select seedlings with basically the same growth vigor, cut off their roots, wash, dry, cut into pieces and mix well;

[0145] (4) Weigh 0.5 g of the sample and place it in a 2 mL centrifuge tube, add 1 steel bead and 1 mL of pre-cooled extraction solution in advance, with 3 replicates for each sample;

[0146] (5) Put the centrifuge tube containing the sample into the sample grinder, shake at 60 Hz for 90 s to homogenize the sample;

[0147] (6) Centrifuge at 12000 g for 10 min at 4 °C, take the supernatant and place it in a new 1.5 mL centrifuge tube, and keep it on ice for later use;

[0148] Set up standard wells, blank wells, measurement wells and control wells, and add the solutions to the 96-well enzyme-linked immunosorbent assay (ELISA) plate in the order from top to bottom in Table 2, and pay attention to avoiding the generation of bubbles;

[0149] Table 2 Reaction system

[0150]

[0151]

[0152] (7) Mix well, react for 30 min at 37 °C in the dark, and measure the absorbance value A at a wavelength of 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader preheated for more than 30 min; ΔA = A 测定 - A 对照 (Make a self-control for each sample);

[0153] (8) Take the concentration of α-ketoglutaric acid in the standard wells as the abscissa and the corresponding A 标准 as the ordinate to plot the standard curve, and calculate the content of α-ketoglutaric acid in the sample according to the standard curve. The calculation formula is as follows.

[0154] Content of α-ketoglutaric acid (μg / g) = (C 标准 × V 标准 ) × ΔA ÷ (A 标准 - A blank) ÷ (W × V1 ÷ V) × Mr × D

[0155] C 标准 : Concentration of the standard sample; V: Volume of the added extraction solution; V1: Volume of the sample to be measured added to the reaction system; V 标准 : Volume of the standard sample added to the reaction system; W: Mass of the sample to be measured; Mr: Molecular weight of α-ketoglutaric acid; D: Dilution factor.

[0156] The detection results are as Figure 9 shown. It can be seen that compared with the wild type, the content of α-ketoglutaric acid in the roots of mutant plants is significantly increased.

[0157] 3.2 Determination of Glutamate Synthase (GOGAT) Enzyme Activity

[0158] The activity of glutamate synthase (GOGAT) was determined using the Glutamate Synthase (GOGAT) Activity Detection Kit from Solarbio. The method was referred to the one provided by the kit with slight modification. The specific method is as follows. All reagents mentioned are from the kit:

[0159] (1) Prepare the working solution according to the kit requirements;

[0160] (2) Among the seedlings hydroponically cultured for 15 days in Example 3.1, select the seedlings with basically the same growth vigor. Cut off their roots, wash, dry, cut into pieces, and mix well;

[0161] (3) Weigh 100 mg of the sample and place it in a 2 mL centrifuge tube containing 1 mL of extraction solution pre-cooled to 4 °C and 1 steel bead. Each sample has 3 replicates;

[0162] (4) Fix the centrifuge tube containing the sample on the homogenizer and shake at 60 Hz for 90 s to homogenize the sample;

[0163] (5) Centrifuge at 10000 g for 10 min at 4 °C, aspirate the supernatant and place it in a new 1.5 mL centrifuge tube, and keep it on ice for further measurement;

[0164] (6) Add samples according to Table 3;

[0165] Table 3 Reaction System

[0166] Component Determination tube (μL) Working solution 180 Sample 20

[0167] (7) Measure the absorbance value at a wavelength of 340 nm using an enzyme-labeled instrument preheated for more than 30 min. The initial absorbance value at 20 s is A1. The enzyme-labeled instrument is controlled at 25 °C for a reaction of 5 minutes, and then measure the absorbance A2 at 5 min 20 s. Calculate ΔA = A1 - A2;

[0168] (8) Calculate the GOGAT enzyme activity according to the following formula:

[0169] GOGAT (U·g-1) = [ΔA × V 反总 ÷ (ε × d) × 109] ÷ (W × V 样 ÷ V 样总 ) ÷ T = 321 × ΔA ÷ W

[0170] V 反总 : Total volume of the reaction system, 2 × 10 -4 L; ε: Molar extinction coefficient of NADH, 6.22 × 103 L·mol -1 ·cm -1; d: optical path of 96-well UV plate, 0.6 cm; V 样 : volume of added sample, 0.02 mL; V 样总 : volume of added extraction solution, 1 mL; T: reaction time, 5 min; W: sample mass, g.

[0171] The detection results are as Figure 10 shown. It can be seen that compared with the wild type, the activity of glutamate synthase in the roots of mutant plants increased significantly.

[0172] The embodiments described above are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. Application of the gene NU4 having the function of regulating nitrogen utilization rate of rice or biological materials related to NU4 in regulating rice growth, characterized in that: The regulating rice growth refers to reducing the protein content of rice seeds and increasing the chalkiness of rice seeds by knocking out the rice nitrogen utilization gene NU4; The nucleotide sequence of the rice nitrogen utilization gene NU4 is shown in SEQ ID NO.1; the amino acid sequence of the protein encoded by the rice nitrogen utilization gene NU4 is shown in SEQ ID NO.2; The biological material related to NU4 is as follows (B1) or (B2): B1) a CRISPR / Cas9-gRNA expression plasmid targeting the rice nitrogen utilization gene NU4; B2) A recombinant microorganism comprising the expression plasmid described in B1).

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