Application of ospth1 gene in improving nitrogen use efficiency and yield of plants

Abstract

The application discloses application of a rice OsPTH1 gene in improving plant nitrogen utilization efficiency and yield increase, and relates to the technical field of genetic engineering.The OsPTH1 mutant plant is obtained through CRISPR-Cas9 technology.Further experiments prove that, compared with wild-type rice, the mutant plant pth1 strain has increased effective tillering, shortened heading stage, increased grain number per panicle, changed dwarf plant height, increased yield per plant and improved nitrogen utilization efficiency.The application proves that the OsPTH1 gene can regulate rice yield and nitrogen utilization efficiency, and lays a foundation for cultivating new rice varieties with high yield and high nitrogen utilization efficiency.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and more specifically to the application of the OsPTH1 gene in improving nitrogen use efficiency and increasing yield in plants. Background Technology

[0002] Rice is one of the main food sources for people in my country and the world, providing the human body with a large amount of dietary energy and nutrients. Therefore, increasing rice yield and nitrogen use efficiency to improve the nutritional quality of rice is an important way to ensure food security and improve the health of residents.

[0003] By utilizing traditional QTL methods and advanced synthetic biology approaches, we aim to discover naturally occurring variation sites, superior haplotypes, and gene combinations for efficient nitrogen use, thereby creating rice genetic materials and planting resources with high nitrogen use efficiency. This has significant theoretical and social value in improving rice nitrogen use efficiency and yield per unit area. Rice nitrogen use efficiency is a quantitative trait regulated by multiple genes, involving physiological processes such as nitrogen absorption, transport, assimilation, and reuse. Previous studies by detecting QTLs and single-gene functionalities related to efficient nitrogen use in rice parents have led to the discovery of highly efficient genes that enhance nitrogen use in rice, including 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.). However, naturally occurring variation sites and superior haplotypes related to efficient nitrogen use still exist in rice, which urgently need to be discovered and applied. Summary of the Invention

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

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] Application of OsPTH1 gene knockout in plant breeding, wherein the application is any one of the following:

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

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

[0009] C. Increase production;

[0010] D. increase effective tillering;

[0011] E. shorten the heading date;

[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 in SEQ ID NO. 2.

[0015] Another object of the present application is to provide the use of the biological material knocking out the above-mentioned OsPTH1 gene in plant breeding, and the biological material is any one of the following:

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

[0017] B. a recombinant vector containing the expression cassette of A;

[0018] C. a recombinant microorganism containing the expression cassette of A or the recombinant vector of B.

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

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

[0021] Another object of the present application is to provide the use of the coding gene of the above-mentioned mutant in plant breeding, and the use is any one of the following:

[0022] A. improving nitrogen utilization efficiency of plants;

[0023] B. increasing grain number per panicle;

[0024] C. increasing yield;

[0025] D. increasing effective tillering;

[0026] E. shortening the heading date;

[0027] F. reducing plant height.

[0028] Another object of the present application is to provide a biological material containing the coding gene of the above-mentioned mutant, and the biological material is an expression cassette, an expression vector, a cloning vector and / or a recombinant microorganism.

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

[0030] A. improving nitrogen utilization efficiency of plants;

[0031] B. increasing grain number per panicle;

[0032] C. increasing yield;

[0033] D. increasing effective tillers;

[0034] E. shortening the heading date;

[0035] F. reducing plant height.

[0036] Another object of the present application is to provide a method for site-directed mutagenesis of OsPTH1 gene using CRISPR / Cas9 system, designing sgRNA sequence based on CRISPR / Cas9 for the target gene, connecting the DNA fragment containing the sgRNA sequence to the vector carrying CRISPR / Cas, transforming rice, and realizing 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 application discloses the application of rice gene OsPTH1 in improving nitrogen utilization efficiency and increasing rice grain length and yield. The present application obtains OsPTH1 mutant plant by CRISPR-Cas9 technology. Further experiments prove that, compared with wild type rice, the mutant plant pth1 strain increases effective tillers, shortens the heading date, increases grain number per panicle, reduces plant height, increases single plant yield, and improves nitrogen utilization efficiency. It is shown that OsPTH1 can regulate rice yield and nitrogen utilization efficiency, and lays a foundation for breeding new rice varieties with high yield and high nitrogen utilization efficiency. BRIEF DESCRIPTION OF DRAWINGS

[0039] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or prior art description will be briefly introduced below. Obviously, the drawings in the following description are only embodiments of the present application, and those skilled in the art can obtain other drawings according to the provided drawings without creative labor.

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

[0041] Figure 2 Figure is the structure of OsPTH1 gene and mutation site and mutation type of mutant.

[0042] Figure 3 Figure is the phenotype of OsPTH1 mutant lines.

[0043] Figure 4 Figure is the statistical data of agronomic traits of OsPTH1 mutant; wherein A is plant height, B is heading date, C is grain number per panicle, D is effective tiller number, and E is yield per plant.

[0044] Figure 5 Figure is the analysis and comparison of nitrogen use efficiency (NUE) of OsPTH1 gene in mutant plants. DETAILED DESCRIPTION

[0045] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative labor fall within the scope of protection of the present application.

[0046] Unless otherwise specified, all technical and scientific terms used herein have the same meanings as generally understood by those skilled in the art to which the present application pertains.

[0047] The experimental methods in the following examples are all conventional methods unless otherwise specified. The experimental materials used in the following examples are all 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 Rice Genome Annotation Project (http: / / rice.plantbiology.msu.edu / ), a suitable target sequence (such as SEQ ID No. 3) is selected, and primers gRNA-OsPTH1-F (such as SEQ ID No. 4) and gRNA-OsPTH1-R (such as SEQ ID No. 5) are designed.

[0050] The primer pair gRNA-OsPTH1-F / R was denatured and annealed to obtain a dimeric product of gRNA-OsPTH1-F / R. The dimeric product of gRNA-OsPTH1-F / R was ligated into the pYLgRNA-U3 vector to obtain an intermediate vector. Then the intermediate vector was ligated into the pYLCRISPR / Cas9P ubi -H final vector (see attached Figure 1 ), to obtain the single-target OsPTH1 CRISPR / Cas9 gene editing vector. After successful construction of the vector, the T0 generation of transgenic plants was finally obtained by Agrobacterium-mediated transformation of callus of Zhonghuang 8015 (ZH8015). The Agrobacterium-mediated rice genetic transformation system was according 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 plant pth1-1 and pth1-2 were obtained (OsPTH1 gene structure and mutation site see attached Figure 2

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

[0052] ATGGCCCCGTCGCCCACCCGCCTCCTCCACCTCCCCCTCCTCGCCCCGAAG

[0053] CCCTCGCCGCCGCGGTGTCTCGTCAGCCGCCGGAGTCGGCCCCGGCCTGC

[0054] CGGCGACGCCGCCCGCTGCGGCTGCGCCACGGAGGCAGGTGGCGGCGGT

[0055] ​GGCGGTGGAGGCTCCGTCGTCGTCGAGGATGATCTCTATGAGCTCCTGCA

[0056] GGTGTCCACTTACCCTCTCTCGTTTCTTTATCTCACCTTCTCAATTCTTTTTT

[0057] TAGCTGCTCATGCATTCCTTGCTTCAATTTTGGAGCTGTTGAATGTTCTTCA

[0058] ATCTTTATCACAATGAAGAAATCAAAGAACATTGCAATAACCAAGTATTA

[0059] TTATTTGACATGGGTTATATTATACTATTCCCTCCGTTTTATATTATAAATC

[0060] GTTTTGATTTTTTTCTTAATCAAACCTTTTTTAAATTTGACAAAGTTTATAG

[0061] AAAAATATAGCACGTCAAAATATATTAAATGTTAGATCTAATAAAATTAA

[0062] TTTAGTTCTATAAATATTGTTATGTTTTTTCTATAAGTTTAGTCAAATTTGC

[0063] AAGAGGTTTGAATTAAAAAAGTCAAAATGACTTATAATACGAAATGGAGG

[0064] GAGTAGTAGATAGCCTCAGCTAGAGAGCTGTTACTGCAACCACCTTTGTT

[0065] GTTGGTAACCTCTCGAATGCTGACCCCAGTTATGTTCATAACCTTTTGAAT

[0066] GGCGAACTCCAGCTTACTATGTCGAATGAAGATGGCTGTGAAAGCTCAAT

[0067] TATACATCACATCCAGTTAACAGGAATCTCTGCACATCTAATGTAAATGA

[0068] GACTGATCTAATAATCAAGCTCTTGATGATTATGCAGTTGATCAATGTTAC

[0069] ATAGGTGGGGAACTGAGGATAACGACTGTTCTGTTTAGTGATGGCAGAGA

[0070] TGGTGATAAACATGTTTAGTTTGCATCATCTGCTCTTAGTCCAGCACTAGC

[0071] TATTAGTGTAAGAAGAGACGAACATCCTACCATAAATTCTTTTAAATAAA

[0072] TTGCTAGGATGGCCATGTTACTGCTCTGAGATGAATTTGTTTCAATAGAAA

[0073] CTAAAATCTTTATGCCCATCGTGTTTCTAAGTGCAAAGCCATTTCTCTCAT

[0074] CCATACTTGATTAGACTGTTGTGTACTAGTAATTTGGTATAAGTGGTTCTT

[0075] GTATGCTAGTTGAGTGACAGAGGGGCACCAATATATCCTTTACAGGTTCT

[0076] ACCAAGAGATTTGCGAGATAATCTGCAGAATGAACCTAGAAAGGATCAG

[0077] CTTTTAGAGGTAAGCTAGATGCTGCTGAGCTCTGTCGTATTTGAATATTCC

[0078] AGTGAAATTAGGCTATTATGGATTTGCAAAAATACCCTAGCTGATGAATG

[0079] GGTGATTAGGTTATTCTGGATTTGGGGAGACGACCTGAGGCACGTTTCCTT

[0080] GGTGACTCTGGTGGCCAATATCTACGGGACAGTGAGGTATAAAGCTACAA

[0081] TAGTACAATTTTGTCCAGTTCACATAGTTATGTTTTGCTTCCTGTGTATTGA

[0082] CAACTAATTTATGTTCTCTGGATTGTTTGTACGATAGATCTCACAGCAAGA

[0083] ATTGGAGGAGGCGCAGAGAGCTGTAGGAGAATTTGGAGGTGACAATCGT

[0084] GCAGGAATCGAAGGTACATTACATAGAATATCTGCCATAAGGAGCAGAA

[0085] AAGGAATGGTTGTTGGTTTAACCTGTCGAGTTGGCCGTGCCGTTACTGGGC

[0086] ATGTTGATATGGTCCGTGATCTCTTAAATTACAAAGAAAGCATTTTGTTTC

[0087] TTGGAAGGTATGTCGCCTTTCGGCTTCAACATTTTTATCATCCTTTTATTAT

[0088] TCCTTTTATTAAATATAAGGTACCCCCAAAATTGCTCAGTACTACTTGCAA

[0089] ACATTACTAGTATTACAAGAGTGACCATATTTTTTTTTAATTCAGCAGCAT

[0090] CTAAAGTAGTCTTAATCTGTGCATAACTGTTCAGCAACACGAGTCTATGAC

[0091] CTTATGTTTTTACTTCCCTTTTCCAGGATATTCTATGAAGTGATATTGTGAT

[0092] GATATTTCAAATGATCAAGCTTTCTATATTTTGCAGAACTAATTAAAGAGG

[0093] AATTAAAGGAAAAGGACCTAAAATTCTCCAGATAAATTCATATAATATAA

[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] TTTTTGTCATTTGCTTATGGATAACAATATGTAGTTAACAGTATGTCTTCG

[0116] AGCATCCTATCCAAATTTCTTACATCTTACATCCTTGCTTTTCGAGAGTTCA

[0117] ATTGCAGATTTGTTTAAATAAACAAAATTTCAGATAAAGGAGCATAATT

[0118] TATGGGCCATTCAGTCATATTGTAATTCTATTTTGTTGGAAAATCAGATTG

[0119] GAGGAGTAGAAACTGTCACTCTTGGTGATGAAGAGGCCCGTGCTCGCCGC

[0120] AGTCAGAAAAGTATTCTGGAGAGGAAGGCCCCTCCAACATTCCCTTTCCT

[0121] TATTGAGATGAGGGAGCGACACTACTGGGTAACTCATCGGGTATGTCTAT

[0122] CAGGCTACAATCTACATATGTCAAACGGTCATGAATATGTAACTTTCAACT

[0123] TCACCTTAATAAGGTGATTTAAGCTGATGCTGAACTAGAATGCTCTGAACT

[0124] GAAAATGAAAACCAAGTAAAAAACATCAGATTTGCACGGAAACAATATA

[0125] TGTCTTAGTTAAGATCCATATATATGTGCTTGCAGGATTCTGTGTAAAGTG

[0126] TAGGAATTGATGGGGGAACCAAATATGATCATCAATTCATCATATTTTGC

[0127] AGTACTAATGCATTTCCTAATTCACAGTTTTACTTATATTGTTCTAACTTGT

[0128] GAACTAGTAGTATTCTTATGTCCTGACTAGGCATGATCAGCAATGATTTTA

[0129] TATCACACAACTGAAATATATATAAACGTTATATTTTTTTTCTTGACCAAA

[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] Rice OsPTH1 gene, the CDS nucleotide sequence 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] Linker primer pair containing target sequence, sequences 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] OsPTH1 mutant plants were obtained by planting in experimental fields at the Fuyang base of the China National Rice Research Institute. Plants were planted individually with a row spacing of 19.8 cm and a plant spacing of 16.5 cm. All field trials were conducted under the same management methods as normal field production. Yield trait assessment: After seed maturity, the middle 15–20 plants in each inner row were selected as the subjects for evaluation, with 10 replicates.

[0190] After multiple generations of cultivation, agronomic traits of the wild type and pth1 lines were investigated and analyzed (see appendix for phenotypes). Figure 3 Statistical data can be found in the appendix. Figure 4 The results showed that, compared with the wild type, the pth1 line was shorter in height and had yellow leaves at maturity. Compared with the wild type ZH8015, the effective tillers and the number of grains per ear were significantly increased in the mutant pth1 line, ultimately leading to a significant increase in yield per plant.

[0191] Example 3: Determination of nitrogen use efficiency in OsPTH1 mutant plants

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

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

[0194] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those 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 invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. Knockout OsPTH1 The application of genes in plant breeding is characterized by, The application is any one of the following: A. Improve the efficiency of nitrogen use in 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 OsPTH1 The nucleotide sequence of the gene is shown in SEQ ID NO.1; The plant in question is rice.

2. The knockout according to claim 1 OsPTH1 The application of genes in plant breeding is characterized by, The OsPTH1 The CDS nucleotide sequence of the gene is shown in SEQ ID NO.

2.

3. Knockout of claim 1 OsPTH1 The application of gene-based biomaterials in plant breeding is characterized by, The biomaterial is any one of the following: A. capable of OsPTH1 Gene knockout expression cassette; B. A recombinant vector containing the expression cassette described in A; C. Recombinant microorganisms containing the expression cassette described in A or the recombinant vector described in B; The plant in question is rice.

Images