Application of rice nitrogen utilization gene NU5 in regulation and control of rice growth

Abstract

The invention discloses application of a rice nitrogen utilization gene NU5 in regulation and control of rice growth, and belongs to the technical field of plant genetic engineering. The nucleotide sequence of the rice nitrogen utilization gene NU5 is as shown in SEQ ID NO. 1; the regulation of rice growth refers to regulation of rice biomass, regulation of rice seed quality and regulation of rice seed protein content. According to the invention, the expression mode of the nitrogen utilization gene NU5 in rice is identified, and a function deletion mutant is obtained by using a gene editing technology. The biomass of rice seedlings of the function deletion mutant is reduced, the thousand seed weight is reduced, the protein content of seeds is reduced, and the nitrogen utilization rate is reduced. The invention provides new gene resources and research basis for rice nitrogen metabolism research, growth regulation mechanism research and seed quality regulation.

Description

Technical Field

[0001] The invention relates to the technical field of plant genetic engineering, and in particular to the application of rice nitrogen utilization gene NU5 in regulating rice growth. Background Art

[0002] Nitrogen is one of the essential nutrients for plant growth and development, and is also an important factor affecting crop yield and quality. As the most important food crop in the world, improving the nitrogen use efficiency (NUE) of rice is of great significance for ensuring food security and achieving sustainable agricultural development. Nitrogen is involved in many important physiological processes in rice, such as photosynthesis, protein synthesis, and hormone regulation. When the nitrogen supply is sufficient, rice plants show faster growth rate, higher biomass accumulation and higher yield; however, excessive application of nitrogen fertilizer not only increases the cost of agricultural production, but also causes significant pollution to the environment, such as eutrophication of water bodies and greenhouse gas emissions.

[0003] Rice nitrogen utilization efficiency is affected by many factors, including nitrogen absorption, transportation, assimilation and redistribution capabilities. At present, studies have discovered a series of genes and regulatory networks related to nitrogen absorption and utilization, such as: nitrogen absorption-related genes: NRT1, NRT2 and other genes encoding nitrate transporters, which are directly involved in nitrate absorption and transportation; nitrogen metabolism-related genes: GS (glutamine synthetase), GOGAT (glutamine-2-oxoglutarate transaminase) and other genes are involved in the nitrogen assimilation process; transcription factor regulatory network: NLP (nitrate-responsive transcription factor) family genes play a key role in regulating the expression of nitrogen-responsive genes. Although a large number of studies have revealed the molecular mechanism of nitrogen utilization, the research and practice of applying these genes to improve rice nitrogen utilization efficiency is still in its infancy, especially the lack of clear and highly practical genes and germplasm resources.

[0004] Improving the nitrogen utilization efficiency of rice through molecular biological methods can significantly reduce the use of nitrogen fertilizers and reduce the negative impact of agricultural production on the environment. However, achieving this goal requires overcoming the following technical bottlenecks: identification and functional verification of key genes, finding genes with important regulatory effects, and in-depth analysis of their functions and mechanisms in the nitrogen utilization process; balancing rice growth and nitrogen utilization, ensuring that the improved genes can both improve nitrogen utilization efficiency and not adversely affect rice yield and quality; the application of efficient breeding technology, combined with gene editing technology (such as CRISPR / Cas9) and modern molecular breeding methods, to achieve the precise introduction and expression regulation of target genes in high-quality rice varieties.

[0005] In summary, discovering new genes for nitrogen utilization in rice and exploring and applying the functions of nitrogen utilization-related genes in rice not only have important scientific research value, but also have broad application prospects in agricultural production practice. Summary of the invention

[0006] The purpose of the present invention is to provide an application of the rice nitrogen utilization gene NU5 in regulating rice growth to solve the problems existing in the above-mentioned prior art. The present invention identifies the expression pattern of the nitrogen utilization gene NU5 in rice and obtains its loss-of-function mutant using gene editing technology. The biomass of the rice seedlings of the loss-of-function mutant is reduced, the thousand-grain weight is reduced, the seed protein content is reduced, and the nitrogen utilization rate is reduced. The present invention provides new gene resources and research basis for the study of rice nitrogen metabolism, the study of growth regulation mechanism and the regulation of seed quality.

[0007] To achieve the above object, the present invention provides the following solutions:

[0008] The present invention provides an application of a rice nitrogen utilization gene NU5 or a related biological material in regulating rice growth, wherein the rice growth regulation refers to reducing rice biomass, rice seed quality and rice seed protein content by knocking out or silencing the rice nitrogen utilization gene NU5; or increasing rice biomass, rice seed quality and rice seed protein content by overexpressing the rice nitrogen utilization gene NU5;

[0009] The nucleotide sequence of the rice nitrogen utilization gene NU5 is shown in SEQ ID NO.1.

[0010] Furthermore, the rice seed quality refers to the thousand-grain weight of rice seeds.

[0011] Furthermore, the amino acid sequence of the protein encoded by the rice nitrogen utilization gene NU5 is shown in SEQ ID NO.2.

[0012] Furthermore, the relevant biological material is any one of the following A1)-A6):

[0013] A1) an expression cassette comprising the rice nitrogen utilization gene NU5;

[0014] A2) a recombinant vector comprising the rice nitrogen utilization gene NU5;

[0015] A3) a recombinant microorganism comprising the rice nitrogen utilization gene NU5;

[0016] A4) a mutant gene of the rice nitrogen utilization gene NU5;

[0017] A5) a CRISPR / Cas9-gRNA expression plasmid targeting the rice nitrogen utilization gene NU5;

[0018] A6) A recombinant microorganism comprising the CRISPR / Cas9-gRNA expression plasmid described in A5).

[0019] Furthermore, the method for regulating rice growth comprises:

[0020] By knocking out the rice nitrogen utilization gene NU5, or introducing the relevant biological material as described in any one of A4) to A6) above into rice plants, the rice biomass, rice seed quality and protein content are reduced;

[0021] Alternatively, the rice nitrogen utilization gene NU5 or the related biological material as described in any one of A1) to A3) above is introduced into rice plants to reduce rice biomass and improve rice seed quality and protein content.

[0022] The present invention also provides a method for regulating rice growth, comprising the steps of knocking out or silencing the rice nitrogen utilization gene NU5, so that the expression amount of the NU5 gene in the rice is reduced, thereby reducing the rice biomass, rice seed quality and protein content; or, by overexpressing the rice nitrogen utilization gene NU5, so that the expression amount of the NU5 gene in the rice is increased, thereby increasing the rice biomass, rice seed quality and protein content;

[0023] The nucleotide sequence of the rice nitrogen utilization gene NU5 is shown in SEQ ID NO.1.

[0024] The present invention also provides a mutant gene of rice nitrogen utilization gene NU5, wherein the mutant gene is NU5-1 or NU5-2;

[0025] The nucleotide sequence of NU5-1 is shown in SEQ ID NO.12; the nucleotide sequence of NU5-2 is shown in SEQ ID NO.13;

[0026] The amino acid sequence of the protein encoded by NU5-1 is shown in SEQ ID NO.14; the amino acid sequence of the protein encoded by NU5-2 is shown in SEQ ID NO.15.

[0027] The present invention discloses the following technical effects:

[0028] The present invention identifies a rice nitrogen utilization gene NU5. After the gene is knocked out, the mutant seedling growth is reduced, the seed 1000-grain weight is reduced, the seed protein content is reduced, and the nitrogen utilization efficiency is reduced. This shows that the NU5 gene plays a key role in regulating rice growth, including rice nitrogen utilization rate, rice yield, rice quality, etc. The present invention provides a new gene resource and research basis for rice nitrogen metabolism research, growth regulation mechanism research and seed quality regulation. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0030] Figure 1 This is a diagram of the spatiotemporal expression pattern of the NU5 gene in different rice tissues;

[0031] Figure 2 The expression pattern of NU5 gene in rice roots in response to nitrogen (N) element;

[0032] Figure 3 This is a map of the CRISPR-Cas9 knockout vector containing the NU5 gene target site sequence;

[0033] Figure 4 Schematic diagram of gene editing of NU5 in the Nipponbare (NIP) background;

[0034] Figure 5 Comparison of plant types of wild-type NIP and mutants nu5-1 and nu5-2 in hydroponic seedlings under low nitrogen (A) and high nitrogen (B) treatments;

[0035] Figure 6 The results of the seedling height (A), fresh weight (B) and dry weight (C) of wild-type NIP and mutants nu5-1 and nu5-2 under different nitrogen treatments are shown in the figure;

[0036] Figure 7 The graph shows the test results of thousand-grain weight (A) and protein content (B) of wild-type NIP mutants nu5-1 and nu5-2 under different nitrogen treatments. DETAILED DESCRIPTION

[0037] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0038] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0039] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0040] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0041] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0042] The nucleotide sequence of the rice nitrogen utilization gene NU5 in the present invention is shown in SEQ ID NO.1:

[0043]

[0044] The amino acid sequence of the protein encoded by the rice nitrogen utilization gene NU5 is shown in SEQ ID NO.2:

[0045] MLRFRGLVHPCSGSQFQFILFQQMLEEKLIDISHFSSSTYNSSYRHNLVLFHLMRSCIATSGTLSPWVKNQSHSSGRFLHFAIIQICWRERKLTLCSTDAIARAKKILSMIPGRATGAYSHSQGIK GLRDEIAAGIASRDGFPANADDIFLTNGASPGVHMMMQLLIRNNRDGIMCPIPQYSLYSASLALHGGALVPYYLDESSGWGLEVSKLKNQLEDARSKGITVRALVVINPGNPTGQILDEQQQYELV KFCKDEELVLLADEVYQENIYVTNKKINSFKKIARSMGYNGDDLQLVSLHSVSKGYYGECGKRGGYMEVTGFSTPVREQLYKIASVNLCSNITGQILASLIMDPPKAGDASYDLYEEEKDNILKSL SRRAKAMESAFNSIDGITCNKTEGAMYLFPRIYLPQKAIEAARAVNKAPDVFYALRLLDTTGIVVTPGSGFGQIIRCYLFTCNSEFLTTQVWSFVKGCRDMAREMHDPAAGGEDTFDDLPLQGIP*.

[0046] Example 1 Exploration of the expression pattern of rice nitrogen utilization gene NU5

[0047] In order to obtain the expression pattern of rice nitrogen utilization gene NU5, RNA was extracted from different tissues (root, stem, leaf, panicle) and seeds at different developmental stages (5, 10, 15, 20, 25, 30 DAF) of rice, and each sample was repeated three times. Among them, the extraction of rice seed RNA was based on the corn endosperm RNA extraction method, using the SDS-TRIZOL method (Joao Leiva, Ricardo Dante and David Holding), and the extraction of RNA from other rice tissues was all done using the TRIZOL method.

[0048] The specific steps are as follows:

[0049] (1) Reagent preparation and sample preparation

[0050] DEPC water was used to prepare SDS-buffer (50mM TRIS pH 8.0, 150mM LiCl, 5mM EDTA pH 8.0, 1% SDS); samples were taken out of the -80°C freezer, placed on ice for later use, the husks were quickly removed with tweezers, and then placed in 1.5mL RNA-free centrifuge tubes placed on ice. Five seeds were taken for samples of each period.

[0051] (2) SDS-buffer extraction

[0052] Add 400 μL of SDS-buffer to the above centrifuge tubes, prepare steel beads that have been scalded on an alcohol flame, add one to each centrifuge tube, and immediately put them into a DNA extraction and proofing shaker to quickly crush the sample for 1 min; add 800 μL of phenol (balanced in TRIS pH 8.0)-chloroform (1:1), shake well, place on ice for 5 min, and centrifuge at 10,000g, 4°C for 10 min; aspirate 500 μL of supernatant and transfer it to a new RNA-Free 1.5 mL centrifuge tube.

[0053] (3) TRIZOL extraction

[0054] Add 1 mL of TRIZOL extract to the sample; add 200 μL of chloroform, shake well, and leave at room temperature for 3 min; centrifuge at 10,000 g, 4°C for 10 min.

[0055] (4) RNA precipitation

[0056] Carefully pipette 500 μL of supernatant into a new RNA-free 1.5 mL centrifuge tube, add 400 μL of isopropanol, mix by inversion, and place on ice for 10 min; centrifuge at 10,000 g, 4°C for 10 min.

[0057] (5) RNA cleaning and dissolution

[0058] Carefully pour out the supernatant, add 500 μL 75% ethanol (prepared with DEPC water), and gently suspend the RNA precipitate at the bottom; centrifuge at 10,000g, 4°C for 5 min, carefully pour out the supernatant, briefly centrifuge for 15 s, use an RNA-free pipette tip to absorb the remaining liquid, and then place in an ultra-clean workbench to air dry; add 40 μL DEPC water and flick to promote the dissolution of the RNA precipitate.

[0059] (6) RNA digestion (removal of gDNA contamination)

[0060] The reaction system consisted of Template RNA 40 μL, 10× Buffer 4.5 μL, and DNase I (RNase-Free) 4 μL. The mixture was treated in a metal bath at 37°C for 30 min, 75°C for 5 min, and stored in a -80°C refrigerator.

[0061] (7) Synthesis of first-strand cDNA

[0062] ReverTraAce qPCR RT Kit (Toyobo, Osaka, Japan) was used for reverse transcription to obtain cDNA. An equal amount of digested RNA was added to each RNase-free centrifuge tube, and RNase-free water was added to mix. The reverse transcription system was as follows: Total RNA (2 μg), RNA-free water was added to 22 μL, reaction conditions: treated in a metal bath at 75°C for 5 minutes, and immediately placed on ice for use; 5×Quantiscript RTBuffer 8 μL, dNTP 4 μL, Oligdt (20) 2 μL, RNA inhibitor 2 μL, ReverTraAce 2 μL were added to each centrifuge tube, and the total volume was 40 μL. The reaction conditions were incubation at 42°C for 60 minutes, incubation at 75°C for 5 minutes to terminate the reaction, and after the reaction was completed, an equal volume of RNA-free water was added to dilute it by one half, and the cDNA template was obtained after mixing, and stored in a -20°C refrigerator for later use.

[0063] (8) Real-time fluorescence quantitative PCR

[0064] The kit used was SYBR Green Real-time PCR Master Mix (Toyobo), and the reaction system was 2×SYBR Premix Ex Taq II 10 μL, 10 μM PCR forward primer 2 μL, 10 μM PCR reverse primer 2 μL, cDNA template 4 μL, and water was added to 20 μL. The PCR program was: 95°C 30 s, 95°C 5 s, 60°C 30 s, and the number of cycles was 40; 95°C 15 s, 60°C 1 min, 95°C 15 s.

[0065] Among them, the PCR primers are: qRT-NU5 F: GCACCTACAACAGCAGCTAC (SEQ ID NO. 3);

[0066] qRT-NU5 R: TCCCGATGTTGCAATACACG (SEQ ID NO. 4).

[0067] (9) Result analysis:

[0068] The results of the real-time PCR experiment are based on 2 -△△CT The method was used for calculation and analysis, and the rice Actin1 (Os03g0718150) gene was used as an internal reference. The primer set of the internal reference gene was: Actin-F: CCCTCCTGAAAGGAAGTACAGTGT (SEQ ID NO.5); Actin-R: GTCCGAAGAATTAGAAGCATTTCC (SEQ ID NO.6).

[0069] The results are as follows Figure 1 As shown, it can be seen that the NU5 gene is highly expressed in the root, panicle and endosperm stages of rice (20DAF).

[0070] Example 2 Prediction of the expression pattern of NU5 gene in rice roots in response to nitrogen treatment

[0071] Using the RAP-DB website (https: / / rapdb.dna.affrc.go.jp), we input the RAP_Locus number of NU5, Os03g0183600, and selected "RXP_5002: Root gene expression profile in response to nutrients" under the "Expression (RiceXPro)" project. Then, we downloaded the data for analysis by selecting nitrogen from the three nutrients "nitrogen (N), phosphorus (P), and potassium (K)". The results showed that the expression level (Cy3 Signal Intensity) of NU5 in the rice root decreased significantly under the "-N" condition for 6 hours and 24 hours ( Figure 2 ), indicating that NU5 is related to the absorption and utilization of nitrogen.

[0072] Example 3 Construction of rice nitrogen utilization gene NU5 knockout vector

[0073] 1. Selection of gRNA target sequence

[0074] According to the CRISPR / Cas9 experimental method, a sequence containing NGG as a recognition site was selected as the knockout target on the second exon of the NU5 gene. The knockout target sequence was 5′-GCAGGGAAACCATCACGGG-3′ (SEQ ID NO. 7), and the PAM sequence was GGG.

[0075] 2. Design of upstream and downstream primers of gRNA oligonucleotide chain

[0076] The upstream primer was NU5_F: 5′-TGTGTGGCAGGGAAACCATCACGGG-3′ (SEQ ID NO. 8);

[0077] The downstream primer is NU5_R: 5′-ACCGTCCCTTTGGTAGTGCCCCAAA-3′ (SEQ ID NO. 9).

[0078] 3. CRISPR / Cas9 vector construction

[0079] The plant Cas9 / gRNA vector construction kit (Catalog.No.BGK032) was used to load the target sequence to form a recombinant vector containing the NU5 gene target ( Figure 3 ). The specific operation method is as follows:

[0080] (1) Formation of oligo dimers

[0081] Take 5 μL of each 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 a double-stranded sequence (oligo dimer) containing the knockout target site.

[0082] (2) Insertion of oligo dimer into vector

[0083] Take 1 μL of Cas9 / gRNA vector, 1 μL of oligo dimer from step (1), 1 μL of buffer 1 and 1 μL of buffer 2, add 6 μL of ddH2O, and react in a 16°C metal bath for 2 hours.

[0084] (3) E. coli transformation

[0085] Take 10 μL of the final product of step (2) and add it to 50 μL of freshly thawed DH5a competent cells, flick to mix, place 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 constant temperature shaker at 37°C, 200 rpm, and after one hour of recovery, spread on a kanamycin-resistant (Kana+) plate.

[0086] (4) Bacterial liquid PCR detection

[0087] The next day, a single clone colony was picked and inoculated into a kanamycin-resistant liquid culture medium, and cultured in a shaker at 37°C until the bacterial solution 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 the plasmid of the positive clone bacterial solution was extracted for later use.

[0088] 4. Agrobacterium transformation and rice genetic transformation

[0089] The successfully constructed positive plasmid was transferred into Agrobacterium (EHA105): 1 μL of plasmid was injected into the freeze-thawed competent Agrobacterium on ice, and then placed on ice for 5 min, liquid nitrogen for 5 min, and 37°C for 5 min, 300 μL of resistance-free LB was added to resuscitate at 28°C for 4 h, and evenly spread on a K+ / Rif resistance (kanamycin + rifampicin) plate. After culturing at 28°C for 3 days, single clones were picked, and positive clones were obtained by detection with hygromycin primers and expanded in 3 mL of liquid K+ / Rif culture medium. The positive K+ / Rif bacterial solution was sent to Wuhan Aidijing Biotechnology Co., Ltd. for rice genetic transformation under the Nipponbare background to obtain the rice NU5 gene knockout strain.

[0090] Example 4 Identification of Rice NU5 Gene Homozygous Knockout Strains

[0091] In order to identify the rice NU5 gene knockout strain obtained in Example 2, the obtained transgenic seedlings were cultured in a normal temperature light incubator for one week, and then the positive seedlings were identified. The specific steps are as follows:

[0092] 1. Knockout transgenic seedling detection

[0093] Twenty T0 transgenic seedlings were obtained, and after being cultured in a normal temperature light incubator for one week, DNA of the 20 seedlings was extracted, and PCR amplification was performed with NU5TF and NU5TR, followed by sequencing. The sequencing results were analyzed, and two transgenic plants with premature termination of protein translation were obtained, namely, two homozygous mutants nu5-1 and nu5-2 with deletion and insertion of the NU5 gene, respectively. The nucleotide sequences of the NU5 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.

[0094] NU5TF: 5′-GTGATGAGATTGCTGCTGGG-3′ (SEQ ID NO. 10);

[0095] NU5TR: 5′-TACAAGAGCTCCGCCATGAA-3′ (SEQ ID NO. 11);

[0096] SEQ ID NO.12 (nu5-1):

[0097]

[0098] SEQ ID NO.13(nu5-2):

[0099] ATGCTCAGGTTTCGTGGTTTAGTCCATCCATGTTCTGGAAGTCAATTTCAATTTATTCTTTTTCAGCAAATGCTAGAAGAGAAATTAATAGATATCTCACATTTTTCTTCCAGCACCTACAACAGCAGCTACAGACACAACCTGGTTCTCTTCCATTTGATGAGATCGTGTATTGCAACATCGGGAACCCTCAGTCCTTGGGTCAAAAACCAATCACATTCTTCAGGGAGGTTCTTGCACTTTGCAATCATCCAAATCTGCTGGAGAGAGAGGAAATTAACTCTTTGCAGCACTGATGCTATTGCTCGGGCAAAGAAAATTCTTTCCATGATTCCTGGAAGAGCCACCGGGGCATATAGTCATAGCCAGGGTATCAAGGGACTGCGTGATGAGATTGCTGCTGGGATTGCCTCCCTGATGGTTTCCCTGCAAATGCAGATGATATATTCCTAACTAATGGAGCAAGTCCTGGTGTACACATGATGATGCAGTTGCTGATAAGGAACAACAGAGATGGCATTATGTGTCCAATTCCTCAATACTCATTGTACTCAGCATCCCTAGCACTTCATGGCGGAGCTCTTGTGCCATATTATCTTGATGAATCCTCAGGATGGGGTTTGGAGGTTTCTAAGCTTAAGAATCAACTTGAAGATGCCAGGTCAAAAGGCATAACTGTTAGGGCGTTGGTGGTGATCAATCCTGGAAATCCTACTGGACAGATTCTTGATGAGCAACAGCAATATGAGCTAGTAAAGTTCTGCAAGGACGAGGAACTTGTTCTTCTGGCGGATGAGGTATACCAAGAGAACATTTATGTTACCAACAAGAAGATCAACTCTTTCAAGAAGATAGCAAGATCCATGGGATACAATGGAGACGATTTACAATTAGTATCATTGCATTCTGTTTCTAAAGGATATTACGGAGAGTGTGGCAAGAGAGGCGGTTACATGGAGGTCACTGGCTTCAGCACTCCAGTTAGAGAACAACTCTACAAAATTGCATCTGTTAACTTGTGTTCAAATATCACCGGCCAGATCCTTGCGAGCCTCATAATGGATCCACCAAAGGCTGGGGACGCATCTTATGACCTCTACGAGGAAGAGAAAGACAACATCCTAAAATCTTTATCTCGTCGTGCAAAGGCAATGGAGTCTGCATTTAACAGTATTGATGGAATTACATGCAACAAGACGGAAGGGGCGATGTATCTGTTCCCACGGATTTATCTACCACAGAAGGCAATTGAGGCTGCCAGGGCTGTCAACAAAGCACCTGATGTATTCTACGCTCTACGTCTTCTTGATACCACCGGCATCGTTGTGACTCCTGGATCTGGTTTTGGACAAATCATCAGATGTTATCTGTTCACCTGCAATTCTGAATTCCTGACCACTCAAGTTTGGTCCTTTGTGAAAGGTTGCAGGGACATGGCACGTGAGATGCACGATCCTGCCGCAGGAGGAGAAGATACCTTCGATGATCTCCCGCTTCAGGGAATTCCATGA;

[0100] SEQ ID NO.14(nu5-1):

[0101] MLRFRGLVHPCSGSQFQFILFQQMLEEKLIDISHFSSSTYNSSYRHNLVLFHLMRSCIAT SGTLSPWVKNQSHSSGRFLHFAIIQICWRERKLTLCSTDAIARAKKILSMIPGRATGAYSHSQ GIKGLRDEIAAGIASL*;

[0102] SEQ ID NO.15 (nu5-2):

[0103] MLRFRGLVHPCSGSQFQFILFQQMLEEKLIDISHFSSSTYNSSYRHNLVLFHLMRSCIAT SGTLSPWVKNQSHSSGRFLHFAIIQICWRERKLTLCSTDAIARAKKILSMIPGRATGAYSHSQGIKGLRDEIAAGIASLMVSLQMQMIYS*.

[0104] After obtaining the stable inherited T2 knockout strain, the target site was further sequenced. The sequencing results of the homozygous mutants nu5-1 and nu5-2 are as follows: Figure 4 Homozygous mutants nu5-1 and nu5-2 were cultured until harvest to obtain mature rice seeds.

[0105] Example 5 Identification of nitrogen utilization ability of rice NU5 gene knockout plants at the seedling stage

[0106] The wild-type (NIP) and mutant (nu5-1, nu5-2) plants were subjected to hydroponic experiments with different nitrogen gradients to detect the growth characteristics of the plants under different nitrogen concentrations.

[0107] 1. The hydroponic experimental method is as follows:

[0108] (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 h;

[0109] (2) After soaking, wrap the seeds with a wet towel and place them at 37°C in the dark for 48 h to germinate;

[0110] (3) The formula of the standard nitrogen nutrient solution refers to the recommended standard of the International Rice Research Institute (IRRI). This experiment set two nitrogen concentrations, 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 formula of the standard nitrogen nutrient solution is shown in Table 1;

[0111] (4) After germination, seeds with relatively consistent bud and root lengths were selected and sown into hydroponic boxes and cultured in a 25°C light incubator (12 h light, 12 h dark);

[0112] (5) The hydroponic method is: hydroponic culture for 3 days, standard nitrogen nutrient solution culture for 4 days, and nutrient solution culture with different nitrogen concentrations for 7 days, for a total of 14 days, and the nutrient solution was replaced every 3 days;

[0113] Hydroponics Notes:

[0114] 1) The water temperature should not be too high or too low, generally around 25°C;

[0115] 2) The roots should be kept away from light to prevent algae growth and root respiration loss;

[0116] 3) When cultivating in large water bodies, the pH of the culture solution should be between 5 and 6, and the lowest should not be lower than 4;

[0117] 4) Cultivation in large water bodies requires water circulation to avoid precipitation of trace elements such as iron under low oxygen conditions;

[0118] 5) The culture medium can be replaced every 3-4 weeks in large water bodies and every 3-5 days in small water bodies. If the seedlings turn yellow, nitrogen should be supplemented in time.

[0119] (6) After 14 days of hydroponic culture, 10 representative plants with basically the same growth were randomly selected to examine the seedling height, fresh weight, and dry weight of the seedlings. This was repeated three times.

[0120] Table 1 Hydroponic nutrient solution (recommended standard nitrogen content) formula

[0121]

[0122] 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 15g of EDTA. When prepared into working solution, the pH needs to be adjusted to around 5.5.

[0123] 2. Hydroponic experiment results:

[0124] 2.1 Comparison of plant morphology between wild type (NIP) and mutant plants under no nitrogen (N0) and 4 times nitrogen (N4) treatments Figure 5 shown.

[0125] 2.2 Agronomic traits of wild type (NIP) and mutant plants under no nitrogen (N0) and 4 times nitrogen (N4) treatments Figure 6 As shown. It can be seen that under low nitrogen and high nitrogen conditions, the plant height of the mutant plants was not significantly different from that of the wild-type NIP ( Figure 6The fresh weight and dry weight of the two mutants were significantly lower than those of the wild type ( Figure 6 B and C). The results showed that the NU5 gene mutation may change the nitrogen utilization efficiency of rice and thus lead to a decrease in growth.

[0126] Example 6 Determination of Thousand-Grain Weight and Protein Content of Rice Seeds

[0127] 1. Thousand-grain weight determination

[0128] The stable homozygous mutant plants and wild type (NIP) of the T2 generation were sown in low nitrogen (LN) and high nitrogen (HN) field environments. Urea (containing 46% N) was applied at 75 kg / hm2 in the low nitrogen field. 2 , urea (containing 46% N) 180kg / hm2 for high nitrogen fields 2 , the same amount of phosphorus fertilizer and potassium fertilizer was used, and mature rice seeds were harvested. The mature seeds were placed in a 65℃ oven and dried to constant weight. The mature seeds were analyzed for grain type: 100 mature and full seeds were randomly selected and weighed. Each sample was repeated 3 times, and the average value × 10 was calculated as the final data of the thousand-grain weight of the seeds. Wild-type seeds were used as controls. The analysis results are as follows Figure 7 As shown in A, it can be seen that the thousand-grain weight of the mutant was significantly lower than that of the wild type under low nitrogen and high nitrogen conditions.

[0129] 2. Determination of total protein content in seeds

[0130] First, the nitrogen content of mature seeds of wild type and mutant was measured using SEALAutoAnalyzer 3 rheometer, and then the protein content was calculated according to the nitrogen content data using the following formula:

[0131] Protein content (%) = C0 × 0.1 × 5.95 ÷ (200 × (1-water content)) × 100

[0132] Wherein, C0 is the N content (mg / L) measured by the rheometer; the moisture content of rice flour is generally about 8%, and this embodiment is calculated based on 8%.

[0133] The method for determining the nitrogen content of seeds is as follows:

[0134] (1) Weigh 200 mg of the sample to be tested and place it in a digestive tube, with 3 replicates for each sample;

[0135] (2) Add 5 mL of H2SO4 to the digestive tube and digest at 290°C for 20 min. After boiling, remove the digestive tube and shake it well.

[0136] (3) Continue digestion for 1 h, shaking every 20 min;

[0137] (4) Remove the digestive tube, wait for it to cool to room temperature, add 2 mL of H2O2, and shake well;

[0138] (5) Digest again for 10 min. After the digestion, 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, 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.

[0139] (6) Add ddH2O to the digestive tube cooled to room temperature and make up to 100 mL;

[0140] (7) Shake the sample to be tested before testing. After standing for 20 minutes, take the supernatant of the sample to be tested with a sample cup and test it with SEALAutoAnalyzer 3 rheometer. The nitrogen content of the test data is calculated according to the following formula:

[0141] Nitrogen content (mg / g) = C0 × 0.1 ÷ 0.2

[0142] Where C0 is the N content (mg / L) measured by the rheometer; 0.1 is the volume of the 100mL sample to be tested, converted to 0.1L; 0.2 is the sample mass.

[0143] The protein content test results are as follows Figure 7 As shown in B. It can be seen that the protein content in the mutant seeds was significantly decreased compared with the wild type under low nitrogen and high nitrogen conditions.

[0144] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. Application of rice nitrogen utilization gene NU5 or its related biological materials in regulating rice growth, characterized in that: The regulating rice growth refers to reducing rice biomass, rice seed quality and rice seed protein content by knocking out or silencing rice nitrogen utilization gene NU5; or increasing rice biomass, rice seed quality and rice seed protein content by overexpressing rice nitrogen utilization gene NU5; The nucleotide sequence of the rice nitrogen utilization gene NU5 is shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that: The rice seed quality refers to the thousand-grain weight of rice seeds.

3. The use according to claim 1, characterized in that: The amino acid sequence of the protein encoded by the rice nitrogen utilization gene NU5 is shown in SEQ ID NO.

2.

4. The use according to claim 1, characterized in that: The relevant biological material is any one of the following A1)-A6): A1) an expression cassette comprising the rice nitrogen utilization gene NU5; A2) a recombinant vector comprising the rice nitrogen utilization gene NU5; A3) a recombinant microorganism comprising the rice nitrogen utilization gene NU5; A4) a mutant gene of the rice nitrogen utilization gene NU5; A5) a CRISPR / Cas9-gRNA expression plasmid targeting the rice nitrogen utilization gene NU5; A6) A recombinant microorganism comprising the CRISPR / Cas9-gRNA expression plasmid described in A5).

5. The use according to claim 1, characterized in that: The method for regulating rice growth comprises: By knocking out the rice nitrogen utilization gene NU5, or introducing the relevant biological material as described in any one of A4) to A6) of claim 4 into the rice plant, the rice biomass, rice seed quality and protein content are reduced; Or, the rice nitrogen utilization gene NU5 or the related biological material as described in any one of A1) to A3) of claim 4 is introduced into rice plants to increase rice biomass, rice seed quality and protein content.

6. A method for regulating rice growth, characterized in that: The method comprises the steps of knocking out or silencing the rice nitrogen utilization gene NU5, so that the expression amount of the NU5 gene in the rice is reduced, thereby reducing the rice biomass, rice seed quality and protein content; Or, a step of increasing the expression level of the rice NU5 gene by overexpressing the rice nitrogen utilization gene NU5, thereby increasing the rice biomass, rice seed quality and protein content; The nucleotide sequence of the rice nitrogen utilization gene NU5 is shown in SEQ ID NO.

1.

7. A mutant gene of rice nitrogen utilization gene NU5, characterized in that: The mutant gene is nu5-1 or nu5-2; The nucleotide sequence of nu5-1 is shown in SEQ ID NO.12; the nucleotide sequence of nu5-2 is shown in SEQ ID NO.13; The amino acid sequence of the protein encoded by nu5-1 is shown in SEQ ID NO.14; the amino acid sequence of the protein encoded by nu5-2 is shown in SEQ ID NO.15.

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