Use of osnac24 gene

Abstract

The application discloses application of an OsNAC24 gene and relates to the technical field of plant genetic engineering, wherein the amino acid sequence coded by the OsNAC24 gene is shown as SEQ ID NO. 2. The OsNAC24 gene belongs to the NAC transcription factor family, and through construction of an OsNAC24 gene overexpression plant, compared with a wild type control, the single gene expression amount of the OsNAC24 gene is enhanced, which causes simultaneous increase of rice nitrogen utilization rate, effective tiller number, grain length, yield and other properties, the genetic effect is remarkable, and the application has great application potential and prospect for improvement of rice yield and variety properties, and provides a new gene resource for efficient breeding of rice.

Description

Technical Field

[0001] The present invention relates to the technical field of plant genetic engineering, and more particularly to the application of OsNAC24 gene. Background Art

[0002] Nitrogen fertilizer is one of the fertilizers needed for agricultural production and contributes the most to crop yield.

[0003] Rice is one of the world's most important food crops and my country's largest. Meeting growing rice production while balancing economic and environmental costs has become a pressing challenge. Through research on nitrogen uptake in rice, scientists have cloned numerous nitrogen assimilation genes, including NRT1.1B, NR2, GRF4, MYB61, OsTCP19, OsNAC42, OsNPF61, and OsNLP4, among other nitrogen-efficient genes. Many of these genes have been used in cultivated rice improvement. Molecular design breeding offers precision and efficiency. By integrating multiple technologies, it proposes optimal genotypes that meet breeding goals, as well as parental matching and progeny selection strategies to achieve these targeted genotypes. This approach improves predictability and efficiency in crop breeding.

[0004] Therefore, continuing to discover more nitrogen-efficient genes is of great guiding significance for revealing the molecular genetic basis of efficient nitrogen utilization in rice and improving the efficiency of molecular design breeding. Summary of the Invention

[0005] In view of this, the present invention provides an application of the OsNAC24 gene.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] The application of the OsNAC24 gene is any one of the following:

[0008] A. Increase the number of rice tillers;

[0009] B. Increase rice grain length;

[0010] C. Increase rice yield;

[0011] D. Improve nitrogen utilization efficiency of rice;

[0012] The amino acid sequence encoded by the OsNAC24 gene is shown in SEQ ID NO.2.

[0013] Preferably, the nucleotide sequence of the OsNAC24 gene is shown as SEQ ID NO.1.

[0014] Another object of the present invention is to provide an application of a biomaterial for increasing the expression of the OsNAC24 gene, wherein the application is any one of the following:

[0015] A. Increase the number of rice tillers;

[0016] B. Increase rice grain length;

[0017] C. Increase rice yield;

[0018] D. Improve nitrogen utilization efficiency of rice;

[0019] The biological material is one of the following:

[0020] a. an expression cassette capable of overexpressing the OsNAC24 gene;

[0021] b. a recombinant vector containing the expression cassette;

[0022] c. a recombinant microorganism containing the expression cassette or the recombinant vector described in b;

[0023] The amino acid sequence encoded by the OsNAC24 gene is shown in SEQ ID NO.2.

[0024] Preferably, the nucleotide sequence of the OsNAC24 gene is shown as SEQ ID NO.1.

[0025] Another object of the present invention is to provide a breeding method for improving nitrogen utilization efficiency of rice by increasing the expression level of the OsNAC24 gene through transgenic means;

[0026] The amino acid sequence encoded by the OsNAC24 gene is shown in SEQ ID NO.2.

[0027] Preferably, the nucleotide sequence of the OsNAC24 gene is shown as SEQ ID NO.1.

[0028] Another object of the present invention is to provide a breeding method for increasing rice yield by utilizing transgenic means to increase the expression level of the OsNAC24 gene;

[0029] The amino acid sequence encoded by the OsNAC24 gene is shown in SEQ ID NO.2.

[0030] Preferably, the nucleotide sequence of the OsNAC24 gene is shown as SEQ ID NO.1.

[0031] Another object of the present invention is to provide a breeding method for increasing rice grain length by using transgenic means to increase the expression level of the OsNAC24 gene;

[0032] The amino acid sequence encoded by the OsNAC24 gene is shown in SEQ ID NO.2.

[0033] Preferably, the nucleotide sequence of the OsNAC24 gene is shown as SEQ ID NO.1.

[0034] Another object of the present invention is to provide a breeding method for increasing the number of rice tillers by utilizing transgenic means to increase the expression level of the OsNAC24 gene;

[0035] The amino acid sequence encoded by the OsNAC24 gene is shown in SEQ ID NO.2.

[0036] Preferably, the nucleotide sequence of the OsNAC24 gene is shown as SEQ ID NO.1.

[0037] Beneficial Effects: The present invention provides applications of the OsNAC24 gene. OsNAC24, a member of the NAC transcription factor family, enhances single-gene expression, leading to simultaneous improvements in rice traits such as nitrogen utilization efficiency, effective tiller number, and yield. This significant genetic effect has enormous potential and prospects for improving rice yield and varietal traits, providing a new genetic resource for nitrogen-efficient rice breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are merely embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying any creative work.

[0039] Figure 1 The accompanying drawings are plasmid maps of the overexpression vector pU1301 provided by the present invention; wherein A is a linear schematic diagram of the vector; and B is a circular schematic diagram of the vector.

[0040] Figure 2 The accompanying figure shows a comparison of the expression levels of the OsNAC24 gene in transgenic plants overexpressing OsNAC24 in the Nipponbare background provided by the present invention. OE-OsNAC24-1 and OE-OsNAC24-2 represent the plant numbers of the two transgenic complementation events, respectively. The same explanation applies to the legend of the following figures.

[0041] Figure 3 The accompanying drawings are phenotypic diagrams of transgenic plants overexpressing OsNAC24 in a Nipponbare background provided by the present invention, as well as statistical diagrams of effective tillering, grain shape, 1000-grain weight, yield per plant, and nitrogen utilization.

[0042] Figure 4The accompanying drawings are phenotype diagrams of transgenic plants overexpressing OsNAC24 in the Nanjing 46 background provided by the present invention, as well as statistical diagrams of effective tillering, grain shape, 1000-grain weight, yield per plant and nitrogen utilization. DETAILED DESCRIPTION

[0043] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. 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 making creative efforts are within the scope of protection of the present invention.

[0044] Example 1 Construction of Rice OsNAC24 Gene Overexpression Plants

[0045] 1. Rice Total RNA Extraction and Reverse Transcription

[0046] Place fresh rice seed samples in an RNase-free centrifuge tube and immediately plunge into liquid nitrogen. Grind the sample into a powder using a grinding rod. Add 300 μL of SDS RNA extraction buffer (5 mM EDTA, 150 mM LiCl, 50 mM Tris-HCl, pH 8.0, 1% SDS) and vortex thoroughly. Add a 1:1 volume ratio of phenol (pH 8.0) to chloroform and vortex for 30 seconds to mix thoroughly. Centrifuge at 12,000 rpm at 4°C for 8 minutes. Pipette the supernatant into a new 1.5 mL RNase-free centrifuge tube, add 1 mL of Trizol, vortex thoroughly, and let stand at room temperature for 10 minutes. Add 200 μL of chloroform and mix thoroughly. Centrifuge at 12,000 rpm at 4°C for 10 minutes. Pipette the supernatant into a new 1.5 mL RNase-free centrifuge tube and add an equal volume of isopropanol to each tube. Invert to mix thoroughly. The sample was centrifuged at 12,000 rpm for 10 minutes at 4°C. The supernatant was discarded and the pellet was washed with 75% ethanol (prepared with DEPC / H2O). The sample was treated with RNase-free DNase I (Takara) at 37°C for 30 minutes. After repeated precipitation with 70% ethanol, the pellet was dried and RNA was dissolved in DEPC / H2O. cDNA (complementary DNA) was synthesized using a TOYOBO reverse transcription kit. 1 μg of RNA was reverse transcribed and stored at -20°C after the reaction.

[0047] 2. Cloning of Rice OsNAC24 Gene and Construction of Overexpression Vector

[0048] According to the OsNAC24 gene (LOC_Os05g34310) sequence published on Rice Genome Annotation Project (http: / / rice.plantbiology.msu.edu / ), a pair of PCR specific primers with restriction endonuclease Kpnl and BamHI joint (OsNAC24F: cggGGTACCATGGCAATGCAGCTGT; OsNAC24 R: cgcGGATCCTCACTCCTGGGGTTG) were designed, and the cDNA of rice variety Nipponbare was used as a template for PCR amplification. The reaction system was as follows: 2x TaqMix 25 μL, cDNA 1 μL, primer-F / R 1 μL, and water to 50 μL. The PCR reaction program was as follows: 95 °C for 3 min, 95 °C for 30 sec, 55 °C for 30 sec, 72 °C for 60 sec, 72 °C for 5 min, 10 °C for 5 min, 35 cycles. Finally, the cDNA full length of the target gene from the start codon to the stop codon was obtained. The obtained gene OsNAC24 sequence was constructed into the pU1301 expression vector by homologous recombination (the plasmid map is shown in Figure 1). Figure 1 ).

[0049] OsNAC24 gene CDS sequence:

[0050] ATGGCAATGCAGCTGTCCTTGCCTGTCTTGCCAACGGGTTTTCGTTTCCATCCAACCGATGAGGAACTAGTCATCAACTACCTCCAGAGGCGTGCTACCGGGCTATCGTGCCCCATCCCCATAATCGCAGATGTCGAAATATACAACTTCAACCCGTGGGAGCTTCCATCCATGGCTCTATTTGGGGAACATGAGTGGTACTTTTTTACACTACGCGACCACAGGTACCCCAATAGTGTGCGCCCTAGTCGCTCAGCGGCGTCAGGCTTTTGGAAGGCCACCGGCACTGACAAGCCCGTCCAAGTTGCTAACATGCAAAGCACTCCTGTAGCTATGAAGAAGGCACTTGTATTCTATGTTGGCCGCCCGCCCATGGAAACCAAGACTACATGGATCATGCATGAGTACCGTCTCACAAACACGGGAGGGTCCACTGCCTCCCACCCATCCTTGTCTTCATCCACCGCACACCCTTCTGTGAAGCTAGACGAGTGGGTGCTATGCAAGATCTTCAACAAGTCCCCAGAGCCGGACAACACCGCACCACCATCAAACGTCGTCTCACGGTTACAGTGCTCGCCGCCGCTGCCGCCGCCGGCGGCACCGCCCGGCAACTACCCGCCGCTGCCGGTGGGTGCGACGAACGACGGCGGCGTGTTCGCCGGCGCCGGCGACATGCTCTTCACCATCCAAGAGCACCAGGAGGGAACACCATCGATGCTGCCGCCGATCCCTAACCTTGAGCCACCGGCGGCAACAATTGGGAATTCCTCTCTCAATGGCACTGCTGCTGCTGCTGCTGCTGCTGATGGCCATGGCCGCCTTGAGGAAGAGGACACCAGCGCCTACACCTTCACCGACCAGGAAATGGAGCAGATGCTCATGGACCTGATGGACCAGGATTTCTTTGGCAATGATCAACCCCAGGAGTGA,如SEQ ID NO.1。

[0051] OsNAC24氨基酸序列为:

[0052] MAMQLSLPVLPTGFRFHPTDEELVINYLQRRATGLSCPIPIIADVEIYNFNPWELPSMALFGEHEWYFFTLRDHRYPNSVRPSRSAASGFWKATGTDKPVQVANMQSTPVAMKKALVFYVGRPPMETKTTWIMHEYRLTNTGGSTASHPSLSSSTAHP SEQ ID NO.2.

[0053] 3. Obtaining OsNAC24 gene overexpressing plants

[0054] The resulting 1301-pUbi::OsNAC24 expression vector was transformed into rice via Agrobacterium-mediated transformation. The specific steps are as follows: 10 μL of the 1301-pUbi::OsNAC24 plasmid was added to 100 μL of competent Agrobacterium and gently mixed. The culture was placed on ice for 5 minutes, then quickly frozen in liquid nitrogen for 5 minutes, and finally incubated in a 37°C waterbath for 5 minutes. The culture was then returned to ice for 2 minutes, followed by the addition of 1 mL of LB liquid medium and incubated on a shaker at 28°C for 2-4 hours. The culture was centrifuged at 8000 rpm for 1 minute at room temperature, the supernatant discarded, and 100-200 μL of the pellet was used to resuspend the pellet. The pellet was then plated on LB medium (100 mg / L Kan, 125 mg / L Rif) and incubated at 28°C for 2-3 days. Once a single colony emerged, positive engineered bacteria were screened and identified. Finally, the callus tissue of Nipponbare was transformed by Agrobacterium-mediated transformation to obtain T0 transgenic plants. The expression level of OsNAC24 gene was determined. The results showed that the expression level of transgenic plants was significantly higher than that of wild type (see Appendix). Figure 2 The Agrobacterium-mediated rice genetic transformation system was based on 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).

[0055] The same method was used to obtain transgenic plants overexpressing OsNAC24 in Nanjing 46 background.

[0056] Example 2 Identification of Nitrogen Utilization Efficiency in OsNAC24 Overexpressing Plants

[0057] The overexpressing plants obtained in this project and their wild-type controls were planted in high nitrogen fields (HN: nitrogen fertilizer application rate of 20 kg / mu) and low nitrogen fields (LN: nitrogen fertilizer application rate of 10 kg / mu) at the China National Rice Research Institute. After the materials matured, the seeds were harvested and cleaned to weigh the yield per plant. The yield per plant was divided by the average nitrogen application rate of the high nitrogen field and the low nitrogen field, respectively, to obtain the nitrogen use efficiency of each genetic material in the high nitrogen field and the low nitrogen field. Compared with the wild type, the nitrogen use efficiency of the OsNAC24 overexpressing plants in the high nitrogen field and the low nitrogen field was significantly improved (see Appendix). Figure 3 GH and attached Figure 4 (GH).

[0058] Example 3 Comparison of agronomic traits of OsNAC24 overexpressing strains

[0059] All materials were planted in the experimental fields of the Fuyang Base of the China National Rice Research Institute in Hangzhou, Zhejiang Province. Individual plants were planted with a row spacing of 19.8 cm and a plant spacing of 16.5 cm. All field experiments were managed uniformly according to normal field production methods. Yield trait assessment: After rice maturity, 10 plants from the 12 OsNAC24 overexpressing lines were harvested. The grains were naturally dried and then stored at room temperature for at least 3 months to ensure grain dryness and relatively consistent moisture content among the lines. The number of effective panicles, grain shape, 1000-grain weight, and yield per plant were evaluated. The number of panicles per plant was calculated by dividing the total number of effective panicles by the number of plants. The number of filled grains per panicle was calculated by dividing the total number of filled grains by the total number of panicles. The 1000-grain weight was calculated by converting the weight of two 300-filled kernels. The yield per plant was calculated by dividing the weight of all filled kernels by the number of plants. Compared with the wild type, the OsNAC24 overexpressing plants showed significantly increased effective tillering, grain length, 1000-grain weight, and yield (see Appendix). Figure 3 AF and attached Figure 4 Medium AF).

[0060] 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.

[0061] The above description of the disclosed embodiments is intended to enable one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily 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 is not limited to the embodiments shown herein but is intended to conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. The application of OsNAC24 gene is characterized by: The application is any of the following: A. Increase the number of rice tillers; B. Increase rice grain length; C. Increase rice yield; D. Improve nitrogen utilization efficiency of rice; The amino acid sequence encoded by the OsNAC24 gene is shown in SEQ ID NO.

2.

2. The use of the OsNAC24 gene according to claim 1, characterized in that: The nucleotide sequence of the OsNAC24 gene is shown in SEQ ID NO.

1.

3. Application of a biomaterial for increasing the expression of the OsNAC24 gene, characterized in that: The application is any of the following: A. Increase the number of rice tillers; B. Increase rice grain length; C. Increase rice yield; D. Improve nitrogen utilization efficiency of rice; The biological material is one of the following: a. an expression cassette capable of overexpressing the OsNAC24 gene; b. a recombinant vector containing the expression cassette; c. a recombinant microorganism containing the expression cassette or the recombinant vector described in b; The amino acid sequence encoded by the OsNAC24 gene is shown in SEQ ID NO.

2.

4. The use of the biomaterial for increasing the expression of the OsNAC24 gene according to claim 3, characterized in that: The nucleotide sequence of the OsNAC24 gene is shown in SEQ ID NO.

1.

5. A breeding method for improving nitrogen utilization efficiency of rice, characterized in that: Using transgenic methods to increase the expression of the OsNAC24 gene; The amino acid sequence encoded by the OsNAC24 gene is shown in SEQ ID NO.

2.

6. A breeding method for increasing rice yield, characterized in that: Using transgenic methods to increase the expression of the OsNAC24 gene; The amino acid sequence encoded by the OsNAC24 gene is shown in SEQ ID NO.

2.

7. A breeding method for increasing rice grain length, characterized in that: Using transgenic methods to increase the expression of the OsNAC24 gene; The amino acid sequence encoded by the OsNAC24 gene is shown in SEQ ID NO.

2.

8. A breeding method for increasing the number of rice tillers, characterized in that: Using transgenic methods to increase the expression of the OsNAC24 gene; The amino acid sequence encoded by the OsNAC24 gene is shown in SEQ ID NO.2.

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