Rice OsZFP12 gene and application of protein coded by rice OsZFP12 gene in rice nitrogen utilization

Abstract

The invention relates to a rice gene OsZFP12 for improving nitrogen utilization efficiency under a low-nitrogen condition and application of an encoding protein of the rice gene OsZFP12, and belongs to the technical field of plant genetic engineering. The spatiotemporal expression mode of the OsZFP12 gene in response to a nitrogen signal is identified, a function knockout plant of the OsZFP12 gene is constructed through a gene editing technology, phenotypic analysis shows that the yield of a single plant and the nitrogen utilization efficiency of a function-deleted rice plant are reduced, and it is indicated that the OsZFP12 regulates and controls the nitrogen utilization efficiency of rice. Further, the gene OsZFP12 provided by the invention is used for constructing a plant overexpression vector, and transgenic rice obtained by carrying out rice transgenic operation can obviously improve the single-plant yield and nitrogen utilization efficiency of the rice under a low-nitrogen condition, which indicates that the implementation of the gene OsZFP12 provided by the invention can create novel rice capable of efficiently utilizing nitrogen; candidate genes can be provided for subsequent crop variety improvement, and great significance is achieved for agricultural production in China.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, and in particular to the application of the rice OsZFP12 gene and its encoded protein in nitrogen utilization in rice. Background Technology

[0002] Nitrogen is one of the essential macronutrients for the normal growth and development of plants. It is not only a component of the synthesis of amino acids and proteins in plants, but also a constituent of important components such as hormones, chlorophyll, and coenzymes. In agricultural production, to meet the urgent demand for food from a growing population, the large-scale application of inorganic nitrogen fertilizer has become a major way to increase crop yields. Rice is one of my country's main food crops. To increase rice yield, the intensity of nitrogen fertilizer application has been increasing year by year. However, the absorption and utilization efficiency of nitrogen fertilizer by rice is generally low, with the actual utilization rate usually less than 50% of the applied amount. A large amount of nitrogen fertilizer remains in the environment without being effectively assimilated by the plants, causing not only resource waste but also adverse environmental impacts, including eutrophication of water bodies, soil acidification, and the emission of nitrous oxide, a greenhouse gas from agriculture. Therefore, improving the nitrogen fertilizer utilization efficiency of rice is an urgent problem to be solved in the development of green and ecological agriculture.

[0003] Currently, some genes related to the nitrogen signaling pathway in rice have been cloned, including nitrate transport genes, ammonium transport genes, and transcription factors. However, nitrogen use efficiency is a complex phenomenon involving multiple biological processes such as nitrogen absorption, transport, assimilation, retransport, and plant growth and development. A comprehensive exploration of the molecular regulatory network of nitrogen absorption and utilization in rice, addressing this complexity, will help identify new ways to improve nitrogen use efficiency in rice and provide a theoretical basis and genetic resources for breeding nitrogen-efficient varieties. Summary of the Invention

[0004] In view of this, the purpose of this invention is to provide an application of the rice OsZFP12 gene to improve nitrogen use efficiency under low-nitrogen conditions, thereby addressing the problems existing in rice nitrogen use. This invention identified the expression pattern of the OsZFP12 gene in rice and obtained loss-of-function mutants and overexpression transgenic plants using gene editing and overexpression technologies, respectively. The loss-of-function mutant rice showed decreased yield and nitrogen use efficiency per plant under low-nitrogen conditions. The OsZFP12 gene overexpression plants showed increased yield and nitrogen use efficiency per plant under low-nitrogen conditions. This invention provides an effective strategy for improving nitrogen use efficiency in rice under low-nitrogen conditions.

[0005] In one aspect, this invention provides the application of the OsZFP12 gene, or the protein it encodes, or biological materials containing the OsZFP12 gene in regulating rice yield per plant and / or nitrogen use efficiency.

[0006] Among them, biological materials may include recombinant DNA, expression cassettes, transposons, plasmid vectors, viral vectors, engineered bacteria or transgenic cells, etc.

[0007] Furthermore, the OsZFP12 gene comprises any of the following nucleotide sequences: (1) The nucleotide sequence as shown in SEQ ID NO:1; (2) A nucleotide sequence that encodes a protein with the same function, obtained by substituting, deleting or inserting one or more nucleotides as shown in (1).

[0008] The nucleotide sequence shown in SEQ ID NO:1 is as follows:

[0009] Furthermore, the protein encoded by the OsZFP12 gene includes any of the following amino acid sequences: (1) The amino acid sequence as shown in SEQ ID NO:2; (2) An amino acid sequence with the same function obtained by replacing, inserting or deleting one or more amino acids as shown in (1).

[0010] The amino acid sequence shown in SEQ ID NO:2 is as follows: MKRFAFEDSDMARVLMLMSSHGQQEQALALPVPVQLPLAAARGDRAPERAFVCKTCNRVFPSFQALGGHRASHKKPRLDGDGDLSLSKPKLHGCSICGLEFAIGQALGGHMRRHRAMTGGMPRAIVVDKKPDVVDVHVHGHDDDGGIKRGGLWLDLNHPPCDDAGDDDAECGHNAAGAGITFHQFLDTGAMAVDCVGY*.

[0011] Furthermore, by knocking out the OsZFP12 gene in rice, or reducing the expression level of the OsZFP12 gene, or reducing the expression level of its encoded protein, or reducing the activity of its encoded protein, the yield per rice plant and / or nitrogen use efficiency can be reduced.

[0012] Furthermore, by overexpressing the OsZFP12 gene in rice, or increasing the expression level of its encoded protein, or increasing the activity of its encoded protein, the yield per rice plant and / or nitrogen use efficiency can be improved.

[0013] In a second aspect, the present invention provides a recombinant overexpression vector comprising the OsZFP12 gene or the protein encoded thereon described above.

[0014] The recombinant overexpression vector used was the pCAMBIA2300 receptor vector, which contains the rice OsZFP12 gene. The vector was obtained by inserting the genomic DNA sequence of the OsZFP12 gene (shown in SEQ ID NO.1) into the plant expression vector pCAMBIA2300 using recombination exchange technology, resulting in the plant expression vector pUBI:OsZFP12-GFP. This overexpression in plants increased rice yield and nitrogen use efficiency.

[0015] In a third aspect, the present invention provides the application of the recombinant overexpression vector in improving nitrogen use efficiency and / or yield per plant in rice under low nitrogen conditions.

[0016] The recombinant overexpression vector can be introduced into the target plant (rice) to increase its yield per plant and / or nitrogen use efficiency; the promoter for gene expression is the strong promoter Ubiquitin, which is used for monocotyledonous plants.

[0017] In a fourth aspect, the present invention provides a method for improving nitrogen use efficiency and yield per plant in rice, including increasing the expression level of the OsZFP12 gene in rice, or increasing the expression level of the protein encoded by it.

[0018] In a fifth aspect, the present invention provides a functional knockout mutant of the OsZFP12 gene, wherein the mutant is oszfp12 #1 or oszfp12 #2, and the mutant can reduce the yield per rice plant and nitrogen use efficiency.

[0019] The nucleotide sequence of oszfp12 #1 is shown in SEQ ID NO.3.

[0020] The amino acid sequence of the protein encoded by oszfp12 #1 is shown in SEQ ID NO.4: MKRFAFEDSVYGARADAHVVARAAGAGAGSAGAGAAAARRRARRPRPGARVRLQDVQPRVPVVPGARRPPCQPQEAEARRRRRPLPVQAQAPRLLHLRPRVRHCARLSAAT*.

[0021] The nucleotide sequence of oszfp12 #2 is shown in SEQ ID NO.5.

[0022] The amino acid sequence of the protein encoded by oszfp12 #2 is shown in SEQ ID NO.6: MKRFAFEDIWRAC*.

[0023] In a fifth aspect, the present invention provides the application of the OsZFP12 gene or the protein encoded thereon in the breeding of high-yielding rice varieties, wherein rice varieties are bred by overexpressing the OsZFP12 gene or increasing the expression level of the protein encoded thereon.

[0024] In a sixth aspect, the present invention provides the application of the OsZFP12 gene or the protein encoded thereon in the breeding of rice varieties that utilize nitrogen efficiently, by overexpressing the OsZFP12 gene or increasing the expression level of the protein encoded thereon.

[0025] The beneficial effects of this invention include at least the following: This invention cloned and identified a rice nitrogen utilization gene, OsZFP12, and demonstrated that this gene sequence can improve the yield per rice plant and nitrogen use efficiency. Therefore, this invention provides a new gene resource and research foundation for rice nitrogen utilization research; this gene can be used to cultivate rice varieties with high nitrogen utilization efficiency and increased yield, effectively improving nitrogen fertilizer use efficiency and reducing the total amount of nitrogen fertilizer applied. This not only saves agricultural resources but also reduces environmental problems such as eutrophication and soil acidification caused by nitrogen fertilizer residues, providing an effective strategy for the development of green and ecological agriculture. Attached Figure Description

[0026] Figure 1 The following diagrams illustrate the expression patterns of the OsZFP12 gene under different treatment conditions: (A) shows the spatiotemporal expression pattern analysis of the OsZFP12 gene in different rice tissues; (B) shows the expression pattern of the OsZFP12 gene in rice roots under nitrogen deficiency treatment; and (C) shows the expression pattern of the OsZFP12 gene in rice roots under NO3 deficiency treatment. - Expression patterns of the OsZFP12 gene at different time points after treatment; (D) shows the expression patterns of the OsZFP12 gene in rice roots at NH4+. + Expression patterns at different times after processing.

[0027] Figure 2 The physical maps of the vectors are shown below; (A) shows the physical map of the overexpression vector pCAMBIA2300-ZFP12; (B) shows the physical map of the CRISPR-Cas9 knockout vector pYLCRISPR / Cas9Pubi-H2-OsZFP12.

[0028] Figure 3This study aims to identify the OsZFP12 gene editing in rice and the phenotype, yield per plant, and nitrogen use efficiency of its mutants under low nitrogen stress. (A) shows a schematic diagram of OsZFP12 gene editing in wild-type (Nipponbare background); (B) shows a schematic diagram of the plant phenotypes of wild-type WT and mutant plants oszfp12 #1 and oszfp12 #2 under low nitrogen conditions; (C) shows a schematic diagram of seeds harvested from a single plant of wild-type and mutant plants oszfp12 #1 and oszfp12 #2 under low nitrogen conditions; (D) shows the yield per plant of wild-type and mutant plants oszfp12 #1 and oszfp12 #2 under low nitrogen conditions; and (E) shows the nitrogen use efficiency of wild-type and mutant plants oszfp12 #1 and oszfp12 #2 under low nitrogen conditions.

[0029] Figure 4 This study aimed to identify OsZFP12 overexpressing lines in rice and analyze their phenotype, yield, and nitrogen use efficiency under low nitrogen conditions. (A) shows the changes in OsZFP12 expression levels in the roots of two independently overexpressing plants (OEZFP #5 and OEZFP #14); (B) shows a schematic diagram of the plant phenotypes of the wild-type and the two independently overexpressing lines (OEZFP #5 and OEZFP #14) under low nitrogen conditions; (C) shows a schematic diagram of seeds harvested from a single plant of the wild-type and the two independently overexpressing lines (OEZFP #5 and OEZFP #14) under low nitrogen conditions; (D) shows the yield statistics of a single plant of the wild-type and the two independently overexpressing lines (OEZFP #5 and OEZFP #14) under low nitrogen conditions; and (E) shows the nitrogen use efficiency statistics of the wild-type and the two independently overexpressing lines (OEZFP #5 and OEZFP #14) under low nitrogen conditions. Detailed Implementation

[0030] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0031] Furthermore, the technical solutions of the various embodiments of the present invention can be combined with each other, but only if they are feasible for those skilled in the art. If the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such combination of technical solutions does not exist and is not within the scope of protection claimed by the present invention.

[0032] The following specific embodiments illustrate the solution proposed in this invention: Example 1 Spatiotemporal expression pattern of rice OsZFP12 gene in response to nitrogen signaling To obtain the spatiotemporal expression pattern of the rice OsZFP12 gene in different tissues, roots from different tissue parts (roots, stems, and leaves) and roots at different growth days (3, 5, 7, 10, 14, and 21 days) were collected. To obtain the expression pattern of the rice OsZFP12 gene in response to nitrogen signals, rice was first hydroponically cultured for 10 days under normal nitrogen conditions, followed by nitrogen deficiency treatment. Root tissues were collected at 0 min, 15 min, 30 min, 1 h, 4 h, and 24 h. After 48 h of nitrogen deficiency treatment, 2.5 mg M KNO3 was added. - Root tissues were treated with NH4Cl and collected at 0 min, 15 min, 30 min, 1 h, 4 h, and 24 h. The sample collected at 0 min served as a control. The collected tissues were ground in liquid nitrogen, and RNA was extracted using a plant RNA extraction kit. cDNA was obtained by reverse transcription using a reverse transcription kit. Real-time quantitative PCR was used to analyze the expression changes of OsZFP12 in different tissues and treatments. The primers for qRT-PCR of the OsZFP12 gene were: OsZFP12 RT-F: AGTTCGCCATTGGCCAGGCTCTC (SEQ ID NO.7); OsZFP12 RT-R: GTGGATGGTTCAGGTCGAGCCACAG (SEQ ID NO.8). The primers for the internal reference gene Ubiquitin were: OsUBI RT-F: AACCAGCTGAGGCCCAAGA (SEQ ID NO.9); OsUBI RT-R: ACGATTGATTTAACCAGTCCATGA (SEQ ID NO.10).

[0033] The results are as follows Figure 1 As shown, Figure 1 A. Quantitative results showed that the OsZFP12 gene was mainly expressed in root tissues; Figure 1 B. Quantitative results showed that the OsZFP12 gene was significantly downregulated under nitrogen-deficient conditions, with the most significant decrease occurring at 1 h, approximately 10-fold. Figure 1 C, Quantitative results showed that the OsZFP12 gene plays a role in NO3. - It was significantly upregulated under conditioned induction, reaching its highest level at 15 min, approximately 30-fold. Figure 1 D, Quantitative results showed that the OsZFP12 gene was present in NH4 + The OsZFP12 gene showed significant upregulation under conditioned induction, reaching its peak at 30 min, approximately 40-fold. This indicates that the OsZFP12 gene can respond rapidly to nitrogen signals.

[0034] Example 2 Obtaining transgenic rice plants with OsZFP12 gene knockout function Using SEQ ID NO:1 as the input sequence on the website http: / / skl.scau.edu.cn / , two pairs of sgRNA sequences were designed in the coding region of OsZFP12, as follows: OsZFP12-sgRNA1: TGCATTCGAGGACAGCGATATGG (SEQ ID NO:11); OsZFP12-sgRNA2-R: CCTCGAGTTCGCCATTGGCC (SEQ ID NO:12). Following the CRISPR / Cas9 vector construction method, the target sequences were loaded to form a recombinant vector containing two OsZFP12-sgRNA target sites. The vector was transformed into *E. coli* DH5α, and the transformation solution was plated on LB agar containing 50 mg / L kanapenem to screen for positive clones. The plasmid was extracted and sequenced for verification. The correctly verified vector was named: pYLCRISPR / Cas9Pubi-H2-OsZFP12 (physical map as shown in 2B). pYLCRISPR / Cas9Pubi-H2-OsZFP12 was transformed into Agrobacterium strain EHA105 using a freeze-thaw method. Positive monoclonal strains containing two OsZFP12-sgRNA target sites were obtained by colony PCR. Positive Kan+ / Rif bacterial cultures were sent to Wuhan Boyuan Biotechnology Co., Ltd. for genetic transformation of rice under the Nipponbare background, resulting in rice OsZFP12 gene knockout lines.

[0035] After culturing 20 T0 generation transgenic seedlings returned by the company in a light incubator for one week, DNA was extracted from the seedlings and amplified by PCR using detection primers. The amplified fragments were sent to the company for next-generation sequencing and compared with the Nipponbare reference sequence to obtain positive single plants with OsZFP12 gene knockout. Simultaneously, seedling DNA was amplified using hygromycin gene detection primers to detect positive single plants that did not contain the Cas9-sgRNA recombinant sequence. oszfp12 #1 and oszfp12 #2 Harvesting, surveying phenotypes.

[0036] The primers for PCR identification of the OsZFP12 gene sequence knockout site are as follows: OsZFP12-jc-F1:ctcacggagtgagcacCAGAGAGGTTGGTTCACAAAG (SEQ ID NO: 13); OsZFP12-jc-R1:ctgagaggctggatggTTGCACGTCTTGCAGACGAACG (SEQ ID NO: 14).

[0037] OsZFP12-jc-F2:ctcacggagtgagcacCACAAGAAGCCGAGGCTCGACG (SEQ ID NO: 15); OsZFP12-jc-R2:ctgagaggctggatggCGACGTCGGGCTTCTTGTCGAC (SEQ ID NO: 16).

[0038] Primers for identifying hygromycin resistance genes: HPTF: CTGCCCGCTGTTCTACAACCGG(SEQ ID NO:17) HPTR: GGAGCATATACGCCCGGAGTC(SEQ ID NO:18) Example 3 Phenotypic analysis of transgenic rice plants with OsZFP12 gene knockout Wild-type plants and homozygous OsZFP12 gene knockout plants (oszfp12 #1, oszfp12 #2) were planted in soil fertilized with 100 kg / ha of urea, and the yield per plant and nitrogen use efficiency were calculated. Results are as follows: Figure 3 As shown, compared with wild-type phenotypic data, it was found that OsZFP12 knockout transgenic plants (oszfp12 #1, oszfp12 #2) suppressed rice yield and nitrogen use efficiency under low nitrogen conditions. Specifically, the yield per plant decreased significantly by about 42% to 50%, and the nitrogen use efficiency decreased significantly by 21% to 33%, indicating that the OsZFP12 gene plays an important role in rice yield and nitrogen use efficiency.

[0039] Example 4 OsZFP12 overexpressing transgenic plants obtained RNA was extracted from the roots of Nipponbare rice seedlings, and cDNA was obtained through reverse transcription. Primers were designed based on the CDS sequence (SEQ ID NO:1) of the OsZFP12 gene to clone the OsZFP12 gene fragment from the Nipponbare rice cDNA. The fragment was then ligated into the basic vector pCAMBIA2300-GFP using the Gateway ligation method. The vector was then transformed into Escherichia coli DH5α, and the transformation solution was plated on LB agar containing 50 mg / L kanapenem to screen for positive clones. Plasmid extraction and sequencing confirmed that the correct vector was the OsZFP12 overexpression vector pUBI:OsZFP12-GFP (physical map as shown in 2A). The amplification primer sequences are: OsZFP12-GFP-F: ctctagagacgtctcgaggaccggtATGAAGAGGTTTGCATTCGA (SEQ ID NO:19); OsZFP12-GFP-R: cctcgcccttgctcaccatggatccGTAGCCGACGCAGTCAAC (SEQ ID NO:20).

[0040] The pUBI:OsZFP12-GFP expression vector was transformed into Agrobacterium competent cells EHA105 using a heat shock method. Positive single-clone strains containing the pUBI:OsZFP12-GFP vector were obtained by colony PCR. These positive strains were then sent to Boyuan Biotechnology Co., Ltd. for transformation to obtain positive T0 generation plants. DNA was extracted from each plant, and PCR amplification was performed using detection primers. Positive single plants were identified by agarose gel electrophoresis. These obtained positive single plants were harvested and propagated until homozygous individuals were reached.

[0041] The primers for PCR identification of positive clones are as follows: OsZFP12-OEjc-F: TAGCCCTGCCTCATACGCT (SEQ ID NO: 21); OsZFP12-OEjc-R:CAGCTTGCCGTAGGTGGCAT (SEQ ID NO:22).

[0042] Example 5 Phenotypic analysis of transgenic rice plants overexpressing the OsZFP12 gene Wild-type plants and homozygous plants overexpressing the OsZFP12 gene (OEZFP #5 and OEZFP #14) were planted in soil fertilized with 100 kg / ha of urea, and the yield per plant and nitrogen use efficiency were recorded.

[0043] As shown in Figure 4, compared with the wild-type phenotypic data, transgenic plants overexpressing the OsZFP12 gene showed improved yield per plant and nitrogen use efficiency under low nitrogen conditions. Specifically, yield per plant increased significantly by about 13% to 19%, and nitrogen use efficiency increased significantly by 18% to 36%. This indicates that the OsZFP12 gene promotes yield per plant and nitrogen use efficiency in rice under low nitrogen conditions and has positive application value for rice germplasm innovation.

[0044] It should be noted that, in this document, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Unless otherwise specified, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes that element.

[0045] The sequence numbers of the above embodiments of the present invention are for descriptive purposes only and do not represent the superiority or inferiority of the embodiments.

[0046] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. Application of the OsZFP12 gene, or the protein it encodes, or biological materials containing the OsZFP12 gene in regulating rice yield per plant and / or nitrogen use efficiency.

2. The application according to claim 1, characterized in that, The OsZFP12 gene includes any of the following nucleotide sequences: (1) The nucleotide sequence as shown in SEQ ID NO:1; (2) A nucleotide sequence that encodes a protein with the same function, obtained by substituting, deleting or inserting one or more nucleotides as shown in (1).

3. The application according to claim 1, characterized in that, The protein encoded by the OsZFP12 gene includes any of the following amino acid sequences: (1) The amino acid sequence as shown in SEQ ID NO:2; (2) An amino acid sequence with the same function obtained by replacing, inserting or deleting one or more amino acids as shown in (1).

4. The application according to claim 1, characterized in that, By knocking out the OsZFP12 gene in rice, or reducing the expression level of the OsZFP12 gene, or reducing the expression level of the protein it encodes, the yield per rice plant and / or nitrogen use efficiency can be reduced.

5. The application according to claim 1, characterized in that, By overexpressing the OsZFP12 gene in rice or increasing the expression level of its encoded protein, the yield per rice plant and / or nitrogen use efficiency can be improved.

6. A recombinant overexpression vector, characterized in that, It includes the OsZFP12 gene as described in claim 1 or the protein encoded thereon.

7. The application of the recombinant overexpression vector according to claim 6 in improving nitrogen use efficiency and / or yield per plant in rice under low nitrogen conditions.

8. A method for improving nitrogen use efficiency and yield per plant in rice, characterized in that, This includes increasing the expression level of the OsZFP12 gene in rice, or increasing the expression level of the protein it encodes.

9. The application of the OsZFP12 gene, or its encoded protein, in the breeding of high-yielding rice varieties, characterized in that... Rice varieties were developed by overexpressing the OsZFP12 gene or increasing the expression level of the protein it encodes.

10. The application of the OsZFP12 gene, or the protein it encodes, in the breeding of nitrogen-efficient rice varieties, characterized in that... Rice varieties were developed by overexpressing the OsZFP12 gene or increasing the expression level of the protein it encodes.

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