Application of OsPTH1 gene in improvement of plant nitrogen utilization efficiency and yield increase

Abstract

The invention discloses application of a rice OsPTH1 gene in improvement of plant nitrogen utilization efficiency and yield increase, and relates to the technical field of gene engineering. According to the invention, an OsPTH1 mutant plant is obtained through a CRISPR-Cas9 technology. Furthermore, experiments prove that compared with wild rice, the mutant plant pth1 strain has the advantages that the effective tillering is increased, the heading period is shortened, the number of grains per ear is increased, the plant height is reduced, the yield of a single plant is increased, and the nitrogen utilization efficiency is improved. The invention proves that the OsPTH1 gene can regulate and control the rice yield and the nitrogen utilization efficiency, and lays a foundation for cultivating a new rice variety with high yield and high nitrogen utilization efficiency.

Description

Technical Field

[0001] The present invention relates to the field of gene engineering technology, and more specifically to the application of OsPTH1 gene in improving plant nitrogen utilization efficiency and increasing yield. Background Art

[0002] As one of the main food sources for people in my country and the world, rice provides a large amount of dietary energy and dietary nutrition for the human body. Therefore, increasing rice yield and increasing nitrogen utilization efficiency to improve the nutritional quality of rice are important ways to ensure food production safety and improve the health of residents.

[0003] Using traditional QTL methods and advanced synthetic biology methods, we can discover natural variation sites, excellent haplotypes and gene combinations for efficient nitrogen utilization, create rice genetic materials and planting resources for efficient nitrogen utilization, and improve rice nitrogen utilization efficiency and yield per unit area, which has important theoretical significance and social value. Rice nitrogen utilization efficiency is a quantitative trait regulated by multiple genes, which involves physiological processes such as nitrogen absorption, transport, assimilation and reuse. In the early stage, scientists have discovered OsNRT1.1B (Hu et al., 2015 Nature Genetics.2. Gao et al., 2019 Nature Communications), OsNRT1.1A (Wang et al., 2018 The Plant Cell), OsNRT2.3A (Fan et al., 2016 PNAS), TOND1 (Zhang et al., 2014 The Plant Journal) and some transcription factors (NAC, etc.) that improve the nitrogen utilization efficiency of rice. However, there are still natural variation sites and excellent haplotypes for efficient nitrogen utilization in rice, which need to be discovered and applied. Summary of the invention

[0004] In view of this, the present invention provides the use of OsPTH1 gene in improving plant nitrogen utilization efficiency and increasing yield.

[0005] In order to achieve the above object, the present invention adopts the following technical solution:

[0006] Application of knocking out the OsPTH1 gene in plant breeding, the application being any one of the following:

[0007] A. Improve the nitrogen utilization efficiency of plants;

[0008] B. Increase the number of grains per ear;

[0009] C. Increase production;

[0010] D. Increase effective tillering;

[0011] E. Shorten the heading period;

[0012] F. Reduce plant height;

[0013] The nucleotide sequence of the OsPTH1 gene is shown in SEQ ID NO.1.

[0014] Preferably, the CDS nucleotide sequence of the OsPTH1 gene is shown as SEQ ID NO.2.

[0015] Another object of the present invention is to provide the use of a biological material in which the OsPTH1 gene is knocked out in plant breeding, wherein the biological material is any one of the following:

[0016] A. Expression cassette capable of knocking out the OsPTH1 gene;

[0017] B. A recombinant vector containing the expression cassette described in A;

[0018] C. A recombinant microorganism containing the expression cassette described in A or the recombinant vector described in B.

[0019] Another object of the present invention is to provide a gene encoding a rice mutant pth1-1, wherein the gene encoding the mutant has a C base deletion at position 81 of the nucleotide sequence shown in SEQ ID NO.1.

[0020] Another object of the present invention is to provide a gene encoding a rice mutant pth1-2, wherein the gene encoding the mutant has a C and G base deletion at positions 79 and 80 of the nucleotide sequence shown in SEQ ID NO.1.

[0021] Another object of the present invention is to provide an application of the coding gene of the above mutant in plant breeding, wherein the application is any one of the following:

[0022] A. Improve the nitrogen utilization efficiency of plants;

[0023] B. Increase the number of grains per ear;

[0024] C. Increase production;

[0025] D. Increase effective tillering;

[0026] E. Shorten the heading period;

[0027] F. Reduce plant height.

[0028] Another object of the present invention is to provide a biological material containing the gene encoding the mutant, wherein the biological material is an expression cassette, an expression vector, a cloning vector and / or a recombinant microorganism.

[0029] Another object of the present invention is to provide the use of the above-mentioned biological material in plant breeding, wherein the use is any one of the following:

[0030] A. Improve the nitrogen utilization efficiency of plants;

[0031] B. Increase the number of grains per ear;

[0032] C. Increase production;

[0033] D. Increase effective tillering;

[0034] E. Shorten the heading period;

[0035] F. Reduce plant height.

[0036] Another object of the present invention is to provide a method for site-directed mutagenesis of the OsPTH1 gene using the CRISPR / Cas9 system, designing a CRISPR / Cas9-based sgRNA sequence for the target gene, connecting a DNA fragment encoding the sgRNA sequence to a vector carrying CRISPR / Cas, and transforming rice to achieve site-directed mutagenesis of rice OsPTH1;

[0037] The nucleotide sequence of the sgRNA action site is shown in SEQ ID NO.3.

[0038] Beneficial effects: The present invention discloses the application of the rice gene OsPTH1 in improving nitrogen utilization efficiency and increasing rice grain length and yield. The present invention obtains OsPTH1 mutant plants by CRISPR-Cas9 technology. It is further proved by experiments that compared with wild-type rice, the mutant plant pth1 strain has increased effective tillering, shortened heading period, increased number of grains per panicle, shorter plant height, increased single plant yield, and improved nitrogen utilization efficiency. It shows that OsPTH1 can regulate rice yield and nitrogen utilization efficiency, laying a foundation for cultivating new rice varieties with high yield and high nitrogen utilization efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] 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 or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0040] Figure 1 The attached figure shows the engineering vector pYLCRISPR / CAS9P ubi -H plasmid map.

[0041] Figure 2 The attached figure shows the structure of OsPTH1 gene, mutation sites and mutation types of mutants.

[0042] Figure 3 The attached figure shows the phenotype of OsPTH1 mutant strains.

[0043] Figure 4 The attached figure shows the statistical data of agronomic traits of the OsPTH1 mutant; wherein A is plant height, B is heading date, C is the number of grains per ear, D is the number of effective tillers, and E is the yield per plant.

[0044] Figure 5 The attached figure shows the analysis and comparison of nitrogen utilization efficiency (NUE) of the OsPTH1 gene in mutant plants. DETAILED DESCRIPTION

[0045] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0046] Unless defined otherwise, 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 belongs.

[0047] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are commercially available products unless otherwise specified.

[0048] Example 1 Construction of CRISPR-Cas9 vector of rice OsPTH1 gene

[0049] According to the OsPTH1 gene sequence (such as SEQ ID No. 1, SEQ ID No. 2) published on the Rice Genome Annotation Project (http: / / rice.plantbiology.msu.edu / ), a suitable target sequence (such as SEQ ID No. 3) was selected, and primers gRNA-OsP TH1-F (such as SEQ ID No. 4) and gRNA-OsPTH1-R (such as SEQ ID No. 5) were designed.

[0050] The primer pair gRNA-OsPTH1-F / R was denatured and annealed to obtain the dimer product of gRNA-OsPTH1-F / R. The dimer product of gRNA-OsPTH1-F / R was connected to the pYLgRNA-U3 vector to obtain an intermediate vector. Then, according to the steps in the reference: Ma X, Zhang Q, Zhu Q, et al. A Robust CRISPR / Cas9 System for Convenient, High Efficiency Multiplex Genome Editing in Monocot and Dicot Plants. Mol Plant. 2015; 8(8): 1274-1284., the intermediate vector was connected to the pYLCRISPR / Cas9P by the side-by-side ligation method. ubi -H terminal vector (see Appendix Figure 1 ) to obtain the single-target OsPTH1 CRISPR / Cas9 gene editing vector. After the vector was successfully constructed, the callus tissue of Zhonghui 8015 (ZH8015) was transformed by Agrobacterium-mediated transformation to obtain T0 transgenic plants. The Agrobacterium-mediated rice genetic transformation system refers to the method reported by Hiei et al. (Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. The Plant Journal, 1994). The OsPTH1 mutant plants pth1-1 and pth1-2 were obtained (the OsPTH1 gene structure and mutation site are shown in the attached Figure 2 ,).

[0051] The nucleotide sequence of rice OsPTH1 gene is as follows:

[0052] ATGGCCCCGTCGCCCACCCGCCTCCTCCACCTCCCCCTCCTCGCCCCGAAG

[0053] CCCTCGCCGCCGCGGTGTCTCGTCAGCCGCCGGAGTCGGCCCCGGCCTGC

[0054] CGGCGACGCCGCCCGCTGCGGCTGCGCCACGGAGGCAGGTGGCGGCGGT

[0055] GGCGGTGGAGGCTCCGTCGTCGTCGAGGATGATCTCTATGAGCTCCTGCA

[0056] GGTGTCCACTTACCCTCTCTCGTTTCTTTATCTCACCTTCTCAATTCTTTTT

[0057] TAGCTGCTCATGCATTCCTTGCTTCAATTTTGGAGCTGTTGAATGTTCTTCA

[0058] ATCTTTATCACAATGAAAATCAAAGAACATTGCAATAACCAAGTATTA

[0059] TTATTTGACATGGGTTATATTATAACTATTCCCTCCGTTTTATATTATAAATC

[0060] GTTTTGATTTTTTTCTTAATCAAACCTTTTTTAAATTGACAAAGTTTATAG

[0061] AAAAAATATGCACGTCAAAATATATTAAATGTTAGATCTAAAAATTAA

[0062] TTTAGTTCTATAAATATTGTTATGTTTTTTCTATAAGTTTAGTCAAATTTGC

[0063] AAGAGGTTTGAATTAAAAAAGTCAAAATGACTTATAATACGAAATGGAGG

[0064] GAGTAGTAGATAGCCTCAGCTAGAGAGCTGTTACTGCAACCACCTTTGTT

[0065] GTTGGTAACCTTCCGAATGCTGACCCCAGTTATGTTCATAACCTTTTGAAAT

[0066] GGCGAACTCCAGCTTACTATGTCGAATGAAGATGGCTGTGAAAGCTCAAT

[0067] TATACATCACATCCAGTTAAACAGGAATCTCTGCCACATCTAATGTAATGA

[0068] GACTGATCTAATAATCAAGCTCTTGATGATTATGCAGTTGATCAATGTTAC

[0069] ATAGGTGGGAACTGAGGATAACGACTGTTCTGTTTAGTGATGGCAGAGA

[0070] TGGTGATAAACATGTTTAGTTTGCATCATCTGCTCTTAGTCCAGCACTAGC

[0071] TATTAGTGTAAGAAGAGACGAACATCCTACCATAAATTCTTTTAAATAAA

[0072] TTGCTAGGATGGCCATGTTACTGCTCTGAGATGAATTGTTTCAATAGAAAA

[0073] CTAAAATCTTTATGCCCATCGTGTTTCTAAGTGCAAAGCCATTTCTCTCAT

[0074] CCATACTTGATTAGACTGTTGTGTACTAGTAATTTGGTATAAGTGGTTCTT

[0075] GTATGCTAGTTGAGTGACAGAGGGGCACCAATATATCCTTTACAGGTTCT

[0076] ACCAAGAGATTTGCGAGATAATCTGCAGAATGAACCTAGAAAGGATCAG

[0077] CTTTTAGAGGTAAGCTAGATGCTGCTGAGCTCTGTCGTATTTGAATATTCC

[0078] AGTGAAATTAGGCTATTATGGATTTGCAAAAAATACCCTAGCTGATGAATG

[0079] GGTGATTAGGTTATTCTGGATTTGGGGAGACGACCTGAGGCACGTTTCCTT

[0080] GGTGACTCTGGTGGCCAATATCTACGGGACAGTGAGGTATAAAGCTACAA

[0081] TAGTACAATTTTGTCCAGTTCACATAGTTATGTTTTGCTTCCTGTGTATTGA

[0082] CAACTAATTTATGTTCTCTGGATTGTTTGTACGATAGATCTCACAGCAAGA

[0083] ATTGGAGGAGGCGCAGAGAGCTGTAGGAGAATTTGGAGGTGACAATCGT

[0084] GCAGGAATCGAAGGTACATTACATAGAATCTGCCATAAGGAGCAGAA

[0085] AAGGAATGGTTGTTGGTTTAACCTGTCGAGTTGGCCGTGCCGTTACTGGGC

[0086] ATGTTGATATGGTCCGTGATCTCTTAAATTACAAAAGCATTTTGTTTC

[0087] TTGGAAGGTATGTCGCCTTTCGGCTTCAACATTTTTATCATCCTTTTATTAT

[0088] TCCTTTTATTAAATAAGGTACCCCCAAATGCTCAGTACTACTTGCAA

[0089] ACATTACTAGTATTACAAGAGTGACCATATTTTTTTTTAATTCAGCAGCAT

[0090] CTAAAGTAGTCTTAATCTGTGCATAACTGTTCAGCAACACGAGTCTATGAC

[0091] CTTATGTTTTTACTTCCCTTTTCCAGGATATTCTATGAAGTGATATTGTGAT

[0092] GATATTTCAAATGATCAAGCTTTCTATATTTTGCAGAACTAATTAAAGAGG

[0093] AATTAAAGGAAAAGGACCTAAAATCTCCAGATAAATTCATAATAATAAA

[0094] CCTTTCTGATAGTCCATGATGTTGAATCAAGATGTTGCAGTTAAGTAAAAT

[0095] GCAACCTCTATGGCTGGAAGAGATCTTCTTTAGACCTTCGATTGTAACTAG

[0096] ATCACTCCTTGGTTAGATTAAGAAATATACCAAGAACCGACATAGTTTGG

[0097] GAGGATTTATCATCATACAAATCTGGAATTCCATGTATGCTGTAGTCTGAT

[0098] TGTTACAAAACACATTTTACATATGCTGTCCACTCATGTCCCTTCAGGGAC

[0099] TCAGGCAAGGTGTGGTGTAGTCATGTGCATTTTAATGCAATTTGAAATTTC

[0100] ATTGATTTGTGTTTTATTTGGCCAGGCCTGGGGTTGGCAAGACTACTGTTA

[0101] TGCGTGAGATTGCACGTGTTCTAGCAGATGAATTTCAGAAAAGAGTGGTA

[0102] ACTAATCGATGTTACAATCTTGCAGACTTTCTTACTCATGTTCACTTTCTTC

[0103] ATATTTAATTCCTTACATGTTCTATCGGTGTATTGATGTTTAGGGCAGGTA

[0104] ATTGTGGATACAAGTAATGAGATTGGTGGGGATGGAGATATTCCTCATGC

[0105] TGCGATTGGTGGTGCAAGAAGAATGCAAGTAGCTCAACCATCAATGCAGC

[0106] ATAGAGTGATGATTGAAGCAGTCGAAAACCATATGCCTGAGGTGGTTATT

[0107] GTAGATGAGATAGGTACTGAAGCAGAGGCGCAAGCTTGTCGATCAATTGC

[0108] AGAAAGGGGTGTGATGCTTATTGGTACAGCTCACGGAGAACATCTCGCAA

[0109] ACATTATAAAGAATCCAACTTTATCTGATTTGGTATGCCATTATTCACTTA

[0110] ATTTGCATTAGATGCATACTGTGTCCTTCTGTTCAATATATAACTTTTACAT

[0111] TGCATCTTACTCAATGCAGAAATGTCAAATGCCTAAAATGAGCTTGACTG

[0112] AGAGTAGTATTCTTTCATTTGCTGTCATCCTATAAGCAGGTTCATTTGCTGT

[0113] ATCTGTAGTTGCAAAAGCCTAGAATTGTTCTGCTATTATTGGTTGAAAATG

[0114] GACTGTGAAGATGACAAGTGCCATTGTCTTATCATGTCATTCAAAGTCATT

[0115] TTTTGTCATTTGCTTATGGATAACAATATGTAGTTAACAGTAGTCTTCG

[0116] AGCATCCTATCCAAATTTCTTACATCTTACATCCTTGCTTTTCGAGAGTTCA

[0117] ATTGCAGATTTGTTTAAATAAACAAAATTTCAGATAAAGGAGCATAATT

[0118] TATGGGCCATTCAGTCATATTGTAATTCTATTTTGTTGGAAAATCAGATTG

[0119] GAGGAGTAGAAACTGTCACTCTTGGTGATGAAGAGGCCCGTGCTCGCCGC

[0120] AGTCAGAAAAGTATTCTGGAGAGGAAGGCCCTCCAACATTCCCTTTCCT

[0121] TATTGAGATGAGGGAGCGACACTACTGGGTACTCATCGGGTATGTCTAT

[0122] CAGGCTACAATCTACATATGTCAAACGGTCATGAATATGTAACTTTCAACT

[0123] TCACCTTAATAAGGTGATTTAAGCTGATGCTGAACTAGAATGCTCTGAACT

[0124] GAAAATGAAAACCAAGTAAAAAACATCAGATTTGCACGGAAACAATATA

[0125] TGTCTTAGTTAAGATCCATATATATGTGCTTGCAGGATTCTGTGTAAAGTG

[0126] TAGGAATTGATGGGGGGAACCAAATATGATCATCAATTCATCATATTTTGC

[0127] AGTACTAATGCATTTCCTAATTCACAGTTTTACTTATATTGTTCTAACTTGT

[0128] GAACTAGTAGTATTCTTATGTCCTGAACTAGGCATGATCAGCAATGATTTTA

[0129] TATCACACAACTGAAATATATAAAACGTTATATTTTTTTTCTTGACCAAA

[0130] TGCTAGGTTAATGAGATAGTTATTTACTATAATGCTAGGTTAATGACTGGT

[0131] TGCCTTTGCGATAATTACTCTTTCTTACATCATCCTTATTGAATTGAACTAC

[0132] TTGTTCACTATTTCTATTGCTGCTCTATTTTTCAACTATTTTTTTTTCAAAGC

[0133] TGAAGATGCTTCCTAAAGGTTATCACCTCTAAATTAATTTTATAGTATTGT

[0134] TCACTGGGCGATGAATACAATACATGGTGTCTGTTTTATGTGCAGACTGAA

[0135] AGGAGTGTGGACATGTTACTTCATGGCAAGAAACCACTTGTTGAGGTAAT

[0136] ATAAAGCTAGCAATTTCATGACGATAAATGCAAGATTTTTTGTGTGTGTAT

[0137] GTGTGTGTAGTTTTCTGTACCACTAGTTAATCGTCTGATTTACCTGTAAGTT

[0138] TCGCTTGCAAGCATAATATTCTATTTGCCATTGTGCTAGCTGCGAACTAGA

[0139] GCATTCCAATGTTGATTCTATGGGTAAAATTATCTAAGTTATTCTTTATTTC

[0140] AGTGCAATGTTGTTCTTTAGCAAAGCAAAATCTTCATTTCTGTAGTGATTC

[0141] TCTCCATGCAATAATTTCAGTCCGAACATAATGTATAAAGCATCAACTGA

[0142] CAACTTGTCACATTTCAGGTCAGGAAAAGGGATAACAAGTTTCAGGTTGT

[0143] CATTGAAAGATGGGCAACGTATGATGGAGATGGACTCTGAAATTCTGAAT

[0144] ATGAGCTTACCTGTTACAGTTGCTTTTATCAAACTGCATGTTCCCGTCCCT

[0145] CAGGATCCCTGTGGACTAATGTATACTTATTCAGCGTGTTGTTGGTATTGA

[0146] TTCCAAAGACTTGTCAAGTTATAGTACTGCTACTCCGGCAAAGGAACTGG

[0147] TGCTGTTCATTGAGGTTAGTGCACAATTTCCATGCTGTTGCTCTCAATCAA

[0148] TTGCATATAAGTTTTTCTTTTCTTTTCCAGTTCAAAATCGTTTTTCTCTGAA

[0149] TCATGATAGGGTTTATTTGTGTAGACTTGAAGTTATAAATGTATTTTCTGTT

[0150] TTGGCACTGAAAATGATTGTGTATTGGTTATATTTCTCATATGGCAGAAAA

[0151] TGAAGTGAAACATCCTGCATAGGTGCCTCTGGTGAACATTTGGACTAACT

[0152] ATCCCGGAATTGTACCTTTTGTGTTTCAATGGCAGTTCATCAATTGATTCCT

[0153] AGTTTCCAGAAATATGAGAGGAAAACAGCAGATAAATCTATCCCAATTAG

[0154] CTAGAAGATTTTACTGTATGAGGTTTTGATTAGCTCAGGAAAATTGTTGTA

[0155] TGGATTCAAAATCTTTGTTGACAATTACATTTGAATACAAAAGGAAGAAG

[0156] TTCAAAAGTAATGCTGTTTAA; SEQ ID No.1.

[0157] The nucleotide sequence of the CDS of the rice OsPTH1 gene is as follows:

[0158] ATGGCCCCGTCGCCCACCCGCCTCCTCCACCTCCCCCTCCTCGCCCCGAAG

[0159] CCCTCGCCGCCGCGGTGTCTCGTCAGCCGCCGGAGTCGGCCCCGGCCTGC

[0160] CGGCGACGCCGCCCGCTGCGGCTGCGCCACGGAGGCAGGTGGCGGCGGT

[0161] GGCGGTGGAGGCTCCGTCGTCGTCGAGGATGATCTCTATGAGCTCCTGCA

[0162] GGTTCTACCAAGAGATTTGCGAGATAATCTGCAGAATGAACCTAGAAAGG

[0163] ATCAGCTTTTAGAGGTTATTCTGGATTTGGGGAGACGACCTGAGGCACGTT

[0164] TCCTTGGTGACTCTGGTGGCCAATATCTACGGGACAGTGAGATCTCACAG

[0165] CAAGAATTGGAGGAGGCGCAGAGAGCTGTAGGAGAATTTGGAGGTGACA

[0166] ATCGTGCAGGAATCGAAGGTACATTACATAGAATATCTGCCATAAGGAGC

[0167] AGAAAAGGAATGGTTGTTGGTTTAACCTGTCGAGTTGGCCGTGCCGTTACT

[0168] GGGCATGTTGATATGGTCCGTGATCTCTTAAATTACAAAGAAAGCATTTTG

[0169] TTTCTTGGAAGGCCTGGGGTTGGCAAGACTACTGTTATGCGTGAGATTGCA

[0170] CGTGTTCTAGCAGATGAATTTCAGAAAAGAGTGGTAATTGTGGATACAAG

[0171] TAATGAGATTGGTGGGGATGGAGATATTCCTCATGCTGCGATTGGTGGTG

[0172] CAAGAAGAATGCAAGTAGCTCAACCATCAATGCAGCATAGAGTGATGATT

[0173] GAAGCAGTCGAAAACCATATGCCTGAGGTGGTTATTGTAGATGAGATAGG

[0174] TACTGAAGCAGAGGCGCAAGCTTGTCGATCAATTGCAGAAAGGGGTGTGA

[0175] TGCTTATTGGTACAGCTCACGGAGAACATCTCGCAAACATTATAAAGAAT

[0176] CCAACTTTATCTGATTTGATTGGAGGAGTAGAAACTGTCACTCTTGGTGAT

[0177] GAAGAGGCCCGTGCTCGCCGCAGTCAGAAAAGTATTCTGGAGAGGAAGG

[0178] CCCCTCCAACATTCCCTTTCCTTATTGAGATGAGGGAGCGACACTACTGGG

[0179] TAACTCATCGGACTGAAAGGAGTGTGGACATGTTACTTCATGGCAAGAAA

[0180] CCACTTGTTGAGGTCAGGAAAAGGGATAACAAGTTTCAGGTTGTCATTGA

[0181] AAGATGGGCAACGTATGATGGAGATGGACTCTGA; SEQ ID No.2。

[0182] PAM

[0183] CRISPR / cas9 target site: GTCTCGTCAGCCGCCGGAGT CGG ; SEQ ID No.3;

[0184] Adapter primer pair containing the target sequence, the sequences are as follows:

[0185] gRNA-OsPTH1-F: gccgTCTCGTCAGCCGCCGGAGT; SEQ ID

[0186] No.4;

[0187] gRNA-OsPTH1-R: aaacACTCCGGCGGTGACGAGA; SEQ ID No. 5.

[0188] Example 2 Phenotypic observation and agronomic trait statistics of OsPTH1 mutant plants

[0189] The OsPTH1 mutant plants obtained were planted in the experimental field of Fuyang Base of China National Rice Research Institute. They were planted individually with a row spacing of 19.8 cm and a plant spacing of 16.5 cm. All field trials were managed in a unified manner in accordance with normal field production methods. Yield trait investigation: After the seeds matured, 15 to 20 plants in the middle of the row were selected as the investigation objects, with 10 replicates.

[0190] After multiple generations of planting, the agronomic traits of the wild type and pth1 lines were investigated and analyzed (see Appendix for phenotypes). Figure 3 , statistical data, see the attached Figure 4 ), the results showed that compared with the wild type, the pth1 strain became shorter in height and had yellow leaves at maturity. Compared with the wild type ZH8015 plants, the effective tillers and the number of grains per ear in the mutant pth1 strain increased significantly, ultimately leading to a significant increase in yield per plant.

[0191] Example 3 Determination of nitrogen utilization efficiency of OsPTH1 mutant plants

[0192] The OsPTH1 mutant plants involved in this project were planted in high nitrogen (HN: nitrogen fertilizer application rate 20kg / mu) and low nitrogen (LN: nitrogen fertilizer application rate 10kg / mu) fields at the China National Rice Research Institute. After the materials matured, the seeds were harvested and cleaned to weigh the single plant yield. The single plant yield was divided by the average nitrogen application rate of low nitrogen fields and high nitrogen fields, respectively, to obtain the nitrogen use efficiency of each genetic material in low nitrogen fields and high nitrogen fields. Compared with the wild type, the nitrogen use efficiency of pth1 plants in low nitrogen fields and high nitrogen fields was significantly improved (see Appendix Figure 5 ).

[0193] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0194] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. Application of knocking out the OsPTH1 gene in plant breeding, characterized in that: The application is any of the following: A. Improve the nitrogen utilization efficiency of plants; B. Increase the number of grains per ear; C. Increase production; D. Increase effective tillering; E. Shorten the heading period; F. Reduce plant height; The nucleotide sequence of the OsPTH1 gene is shown in SEQ ID NO.

1.

2. The use of knocking out the OsPTH1 gene in plant breeding according to claim 1, characterized in that: The CDS nucleotide sequence of the OsPTH1 gene is shown in SEQ ID NO.

2.

3. Use of the biological material with the OsPTH1 gene knocked out according to claim 1 in plant breeding, characterized in that: The biological material is any one of the following: A. Expression cassette capable of knocking out the OsPTH1 gene; B. A recombinant vector containing the expression cassette described in A; C. A recombinant microorganism containing the expression cassette described in A or the recombinant vector described in B.

4. A gene encoding a rice mutant pth1-1, characterized in that: The coding gene of the mutant has a C base deletion at the 81st position of the nucleotide sequence shown in SEQ ID NO.

1.

5. A gene encoding a rice mutant pth1-2, characterized in that: The coding gene of the mutant has a G and C base deletion at positions 79 and 80 of the nucleotide sequence shown in SEQ ID NO.

1.

6. Use of the gene encoding the mutant according to claim 4 or 5 in plant breeding, characterized in that: The application is any of the following: A. Improve the nitrogen utilization efficiency of plants; B. Increase the number of grains per ear; C. Increase production; D. Increase effective tillering; E. Shorten the heading period; F. Reduce plant height.

7. A biological material containing a gene encoding a mutant according to claim 4 or 5, characterized in that: The biological material is an expression cassette, an expression vector, a cloning vector and / or a recombinant microorganism.

8. The use of the biological material according to claim 7 in plant breeding, characterized in that: The application is any of the following: A. Improve the nitrogen utilization efficiency of plants; B. Increase the number of grains per ear; C. Increase production; D. Increase effective tillering; E. Shorten the heading period; F. Reduce plant height.

9. A method for site-directed mutagenesis of the OsPTH1 gene using the CRISPR / Cas9 system, characterized in that: Designing a CRISPR / Cas9-based sgRNA sequence for the target gene, connecting a DNA fragment encoding the sgRNA sequence to a vector carrying CRISPR / Cas, and transforming rice to achieve site-directed mutagenesis of the rice OsPTH1 gene; The nucleotide sequence of the sgRNA action site is shown in SEQ ID NO.3.

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