Application of potassium transporter gene OsHAK25 in improvement of phosphorus absorption capacity of rice

Abstract

The invention provides an application of a potassium transporter gene OsHAK25 in improving the phosphorus absorption capacity of rice, a nucleotide sequence of the OsHAK25 gene is shown as SEQ ID NO.2, and the OsHAK25 gene negatively regulates the phosphorus absorption capacity of the rice. The OsHAK25 gene of rice is knocked out to obtain an oshak25 mutant plant, under phosphorus treatment, the content of inorganic phosphorus in leaves of the oshak25 mutant plant and a wild type plant is measured, it is found that the content of the inorganic phosphorus in the leaves of the oshak25 mutant plant is remarkably higher than that of the wild type plant, and it is indicated that the phosphorus absorption capacity of the oshak25 mutant plant is higher. By analyzing the expression quantity of the phosphorus absorption related genes of the overground parts and root systems of an oshak25 mutant plant and a wild type plant, it is found that the expression quantity of the OsSPX2 genes of the overground parts and root systems of the oshak25 mutant plant is significantly lower than that of the wild type plant. It is shown that an oshak25 mutant plant can increase the expression quantity of phosphorus absorption related genes such as the OsPT2 gene, the OsPT3 gene and the OsPAP21b gene by down-regulating the expression quantity of the OsSPX2 gene, and therefore phosphorus absorption of rice is promoted.

Description

Technical Field

[0001] This invention belongs to the fields of plant breeding and biological genes, specifically involving the application of the potassium transport gene OsHAK25 in improving the phosphorus uptake capacity of rice. Background Technology

[0002] Phosphorus, an essential macronutrient for plant growth and development, is a vital cellular component, including phospholipids, nucleic acids, and proteins, playing a crucial role in energy metabolism, photosynthesis, DNA replication, and intracellular signal transduction. Since most phosphorus in soil exists as organic phosphorus or is fixed by metal oxides and carbonate compounds, the amount of available phosphorus directly usable by plants is typically low. Soil phosphorus deficiency has become one of the major limiting factors in agricultural production. It is estimated that over 50% of the world's arable land is phosphorus deficient, thus agricultural production often relies on the application of large amounts of phosphate fertilizers to ensure crop yields. However, the main source of phosphate fertilizers—phosphate rock—is finite and non-renewable. At the current rate of mining and use, it is estimated that global commercial phosphate rock reserves will be depleted within the next approximately 300–400 years. Therefore, cultivating crops that efficiently absorb and utilize phosphorus is a strategy to reduce phosphate fertilizer application.

[0003] Rice is a major food crop in my country, requiring a large amount of phosphorus and being sensitive to low phosphorus stress. The available phosphorus content in the soil of major rice-growing areas in my country is generally low, leading to a severe decline in rice yield due to phosphorus deficiency. Although some genes have been reported to improve phosphorus uptake in plants, the resources of effective phosphorus-efficient genes that can be readily applied in field production remain very limited. Summary of the Invention

[0004] To address the aforementioned problems, the present invention aims to provide the application of the potassium transporter gene OsHAK25 in improving phosphorus uptake in rice. To achieve this objective, the technical solution adopted by the present invention is as follows:

[0005] The nucleotide sequence of the OsHAK25 gene is shown in SEQ ID NO.2. The OsHAK25 gene negatively regulates the phosphorus uptake capacity of rice.

[0006] Furthermore, the CDS sequence of the OsHAK25 gene is shown in SEQ ID NO.3.

[0007] Furthermore, the amino acid sequence of the protein encoded by the CDS sequence of the OsHAK25 gene is shown in SEQ ID NO.1.

[0008] Furthermore, the OsHAK25 gene was knocked out to obtain the oshak25 mutant plant. The inorganic phosphorus content in the leaves of the oshak25 mutant plant was significantly higher than that of the wild type under phosphorus treatment.

[0009] Furthermore, the expression level of the OsSPX2 gene in the oshak25 mutant plant is lower than that in the wild-type plant, and the expression level of the OsSPX2 gene is negatively correlated with the phosphorus uptake capacity of rice.

[0010] Furthermore, the expression level of the OsSPX2 gene was reduced to increase the expression levels of the OsPT2, OsPT3, and OsPAP21b genes, which are used to positively regulate the phosphorus uptake capacity of rice.

[0011] A breeding method for rice with high phosphorus absorption function involves knocking out the OsHAK25 gene as described above in rice. The method includes:

[0012] Design target primers for sgRNA, wherein the target primers for sgRNA include OsHAK25-U3F and OsHAK25-U3R;

[0013] A knockout vector was constructed, wherein the knockout vector was OsHAK25CRISPR / Cas9;

[0014] The knockout vector was transferred into an Agrobacterium-mediated transformation, and the Agrobacterium-mediated transformation was used to infect rice callus tissue to obtain transgenic rice plants T0.

[0015] The oshak25 mutant plant was screened from the transgenic rice plant T0.

[0016] Furthermore, the target primer sequence of the sgRNA is as follows:

[0017] OsHAK25-U3F: 5'-ggcaGTGAGAGGGCTCGGTACACA-3';

[0018] OsHAK25-U3R: 5'-aaacTGTGTACCGAGCCCTCTCAC-3'.

[0019] Furthermore, the construction of the knockout vector includes:

[0020] The target primers of the sgRNA were annealed into double strands to obtain the target fragment;

[0021] The pYLCRISPR-Cas9-gxy plasmid was digested with BsaI restriction enzyme to obtain the vector pYLCRISPR / Cas9-L;

[0022] The target fragment was ligated to the vector pYLCRISPR / Cas9-L using T4 ligase to obtain the knockout vector OsHAK25CRISPR / Cas9.

[0023] Furthermore, the screening of oshak25 mutant plants from transgenic rice plants T0 includes:

[0024] Primers were designed to target the genomic DNA of transgenic rice plant T0, and target DNA fragments were obtained.

[0025] The target DNA fragment was amplified using PCR technology to obtain the amplified target DNA fragment.

[0026] The amplified target DNA fragments were sequenced to screen out homozygous oshak25 mutant plants that had Cas9 removed.

[0027] Compared with the prior art, the beneficial effects of the present invention include: by knocking out the OsHAK25 gene in rice, oshak25 mutant plants were obtained. The inorganic phosphorus content in the leaves of oshak25 mutant plants and wild-type plants were measured under the phosphorus levels of 1 mM, 0.5 mM and 0.1 mM, respectively. The results showed that the inorganic phosphorus content in the leaves of oshak25 mutant plants was significantly higher than that in wild-type plants at all three phosphorus levels, indicating that oshak25 mutant plants have a stronger phosphorus absorption capacity than wild-type plants. Analysis of the relative expression levels of phosphorus uptake-related genes such as OsSPX2, OsPT2, OsPT3, and OsPAP21b in the aboveground parts and roots of oshak25 mutant and wild-type plants revealed that the expression level of OsSPX2 gene in the aboveground parts and roots of oshak25 mutant plants was significantly lower than that in wild-type plants, while the expression levels of OsPT2, OsPT3, and OsPAP21b genes in the roots of oshak25 mutant plants were significantly higher than those in wild-type plants. This suggests that oshak25 mutant plants may promote phosphorus uptake in rice by downregulating the expression level of OsSPX2 gene and increasing the expression levels of phosphorus uptake-related genes such as OsPT2, OsPT3, and OsPAP21b genes. Attached Figure Description

[0028] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0029] Figure 1 This is an editing type diagram of the oshak25 mutant plant of the present invention, wherein the oshak25 mutant plant includes the oshak25-1 mutant plant and the oshak25-2 mutant plant. White squares represent UTRs, black squares represent exons, gray lines represent introns, and blue squares represent target sites.

[0030] Figure 2 The inorganic phosphorus content in the leaves of the wild-type plants ZH11, the oshak25-1 mutant plants, and the oshak25-2 mutant plants of this invention is shown in the figure. * P<0.05, ** P<0.01, and the t-test was used to analyze the significance of the difference. Each genotype and each treatment contained 4 biological replicates, and FW was fresh weight.

[0031] Figure 3 This invention illustrates the expression of phosphorus absorption-related genes in the aboveground parts and roots of wild-type plants ZH11, oshak25-1 mutant plants, and oshak25-2 mutant plants. Figure A shows the OsSPX2 gene, Figure B shows the OsPT2 gene, Figure C shows the OsPT3 gene, and Figure D shows the OsPAP21b gene. ** P<0.01, and the significance of the difference was analyzed by t test. Each genotype and each tissue contained 3 biological replicates. Detailed Implementation

[0032] The following embodiments are used to illustrate the present invention, but are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of protection of the present invention. Unless otherwise specified, the experimental materials, reagents, instruments, etc., used in the embodiments of the present invention are commercially available; unless otherwise specified, all technical means in the embodiments of the present invention are conventional means well known to those skilled in the art.

[0033] Example 1: Obtaining the Oshak25 mutant rice plant

[0034] The OsHAK25 gene originates from rice (Oryza sativa L.), and OsHAK25 (High-Affinity K) is derived from this gene. + Transporter 25 is a high-affinity potassium (K) molecule in rice. + The OsHAK25 transporter gene, belonging to the HAK / KUP / KT family, is responsible for regulating potassium absorption and transport in rice, thereby affecting its growth, development, and stress resistance. The nucleotide sequence of the OsHAK25 gene, as shown in SEQ ID NO.2, consists of 4952 nucleotides, and the CDS sequence, as shown in SEQ ID NO.3, consists of 2316 nucleotides. The amino acid sequence of the protein encoded by the CDS sequence of the OsHAK25 gene, as shown in SEQ ID NO.1, consists of 771 amino acid residues.

[0035] The oshak25 mutant plants were obtained by knocking out the OsHAK25 gene. The specific steps are as follows:

[0036] (1) Design of sgRNA

[0037] First, the nucleotide sequence of the OsHAK25 gene, as shown in SEQ ID NO.2, was used as a reference sequence. Then, the key exon was determined, and the designed knockout target sequence was located in the fourth exon of the OsHAK25 gene. The specific target sequence is GTGAGAGGGCTCGGTACACA. Finally, a pair of sgRNA primers, including OsHAK25-U3F and OsHAK25-U3R, were designed based on the target sequence.

[0038] The primer sequences are as follows:

[0039] OsHAK25-U3F: 5'-ggcaGTGAGAGGGCTCGGTACACA-3';

[0040] OsHAK25-U3R: 5'-aaacTGTGTACCGAGCCCTCTCAC-3'.

[0041] To facilitate subsequent ligation with the linearized vector, the 5' end of the OsHAK25-U3F primer sequence is fitted with an ggca adapter, and the 5' end of the OsHAK25-U3R primer sequence is fitted with an aaac adapter.

[0042] (2) Constructing a knockout vector

[0043] First, the aforementioned pair of sgRNA primers, OsHAK25-U3F and OsHAK25-U3R, are annealed to form double strands. This is done by mixing equal volumes of OsHAK25-U3F and OsHAK25-U3R, for example, 0.5 μM each, in annealing buffer. The mixture is then heated to 95°C and slowly cooled to room temperature to anneal and form double-stranded DNA. The two ends of this double-stranded DNA are the sticky ends complementary to the ends of the linearized vector, thus completing the preparation of the target fragment. Next, a specific restriction endonuclease, such as BsaI, is used to digest the pYLCRISPR-Cas9-gxy plasmid. After single digestion, the original circular plasmid becomes a linear, terminal DNA fragment, thus obtaining the linearized vector pYLCRISPR / Cas9-L. Then, the target fragment was ligated with the linearized vector pYLCRISPR / Cas9-L using T4 ligase. Specifically, the annealed double-stranded DNA was mixed with the enzyme-digested linearized vector pYLCRISPR / Cas9-L, and the ligation reaction was carried out using T4 DNA ligase to finally obtain the knockout vector OsHAK25CRISPR / Cas9.

[0044] (3) Agrobacterium genetic transformation

[0045] Agrobacterium-mediated transformation is a technique that utilizes the natural ability of Agrobacterium tumefaciens to transfer and integrate specific DNA fragments (T-DNA) into the plant genome. First, using Agrobacterium-mediated transformation, the constructed knockout vector OsHAK25CRISPR / Cas9 is introduced into Agrobacterium tumefaciens strains that have been devirulent but retain vir gene function, such as LBA4404, EHA105, and GV3101, via electroporation or freeze-thaw methods. The strains are then cultured on media containing appropriate antibiotics, and Agrobacterium strains containing the OsHAK25CRISPR / Cas9 are selected for single-cloning.

[0046] Next, plant recipient materials were prepared, specifically plant tissues readily accepting exogenous DNA, i.e., explants. Explants possess vigorous meristem activity and are more likely to accept T-DNA. Commonly used explants include rice callus, induced from mature embryos. Single-cloned Agrobacterium strains were used to infect rice callus, specifically Zhonghua 11 (ZH11) rice. During infection, phenolic compounds such as acetylsuccinone were added, which strongly induce the expression of the Agrobacterium vir gene, thereby efficiently activating T-DNA cleavage and transfer. The infected rice callus was then transferred to a co-culture medium and cultured in the dark at a suitable temperature for 2-3 days. During this process, Agrobacterium attaches to the plant cell wall, the vir gene product cleaves the T-DNA single strand from the vector, and injects it into the plant cell through a process similar to bacterial conjugation. After entering the plant cell nucleus, the T-DNA randomly integrates into the plant's genomic DNA.

[0047] Then, the rice callus tissue is screened and transformed. After co-culture, a large number of Agrobacterium bacteria are present on the surface and inside the rice callus tissue, which need to be removed. Antibiotic-containing culture media are typically used to kill the Agrobacterium bacteria without harming the plant cells. Simultaneously, selection agents such as hygromycin and glufosinate are added to the culture medium. Only plant cells that successfully integrate T-DNA can survive and grow; untransformed cells die. The surviving resistant rice callus tissue is transferred to a regeneration medium containing a specific ratio of plant hormones to induce the rice callus tissue to differentiate into shoots and roots, eventually developing into complete transgenic seedlings, i.e., transgenic rice plants T0.

[0048] (4) Identification of oshak25 mutant plants

[0049] First, genomic DNA was extracted from the transgenic rice plant T0. Primers were designed near the target site of the genomic DNA to obtain the target DNA fragment. The target DNA fragment was amplified by PCR and sequenced to determine whether there was a mutation in the OsHAK25 gene. The positive transgenic rice plant T0 that passed the identification was the oshak25 mutant plant with the OsHAK25 gene knocked out.

[0050] The specific primer sequences for amplification are as follows:

[0051] OsHAK25-Cas9-F: 5'-CAACTTCCTGTGACGTGTGC-3';

[0052] OsHAK25-Cas9-R: 5'-GTTGCACTAGGAGGGGATGG-3'.

[0053] The oshak25 mutant plants with the target gene successfully knocked out were transplanted into soil. After maturation, the seeds were harvested as transgenic rice plants T1. Genomic DNA was extracted from the transgenic rice plants T1, and the target fragment was amplified and sequenced using primers OsHAK25-Cas9-F / OsHAK25-Cas9-R. Then, primers HPT-F / HPT-R were used to identify whether the Cas9 protein sequence was inserted. Finally, homozygous oshak25 mutant plants with Cas9 removed were obtained, including oshak25-1 mutant plants and oshak25-2 mutant plants. Figure 1 As shown, the oshak25-1 mutant plant has an insertion of one base, while the oshak25-2 mutant plant has a deletion of 41 bases. Both mutant plants have caused frameshift mutations.

[0054] The specific HPT-F / HPT-R primer sequences are as follows:

[0055] HPT-F: 5'-AGCTGCGCCGATGGTTTCTACAA-3';

[0056] HPT-R: 5'-ATCGCCTCGCTCCAGTCAATG-3'.

[0057] Example 2: Determination of inorganic phosphorus content in leaves of rice oshak25 mutant

[0058] Kumura Solution is a chemically defined liquid culture medium containing a relatively high concentration of nitrogen source, particularly ammonium nitrogen (NH4). +This matches the physiological needs of crops such as rice, providing a balanced supply of trace elements, such as iron, manganese, zinc, copper, molybdenum, and boron, which are crucial for normal cell growth and differentiation. First, wild-type plants ZH11, oshak25-1 mutants, and oshak25-2 mutants were cultured in Kimura nutrient solution under three phosphorus levels: 1 mM, 0.5 mM, and 0.1 mM. After 6 weeks of culture, leaves from these plants were collected. Next, the inorganic phosphorus content in the leaves was determined using the Malachite Green Method, a highly sensitive and widely used biochemical analysis method for the quantitative detection of inorganic phosphate (Pi). Its core principle is that malachite green dye forms a green complex with phosphate in the presence of molybdate; the concentration of phosphate is quantified by measuring the intensity of the color. The malachite green method is not only extremely sensitive, capable of detecting phosphates at the nanomolar (nmol) or even pmol (pmol) level, but also has the advantages of being simple, quick to operate, and low in reagent cost.

[0059] like Figure 2 As shown, by measuring the inorganic phosphorus content in the leaves of wild-type plants ZH11, oshak25-1 mutant plants, and oshak25-2 mutant plants cultured under the above three phosphorus levels, it was found that the inorganic phosphorus content in the leaves of oshak25-1 mutant plants and oshak25-2 mutant plants was significantly higher than that in wild-type plant ZH11 under the above three phosphorus levels. Specifically, under 1 mM phosphorus level culture conditions, the inorganic phosphorus content in the leaves of wild-type plants ZH11, oshak25-1 mutant plants, and oshak25-2 mutant plants were 4.09 mg / g, 6.08 mg / g, and 4.81 mg / g fresh weight (FW), respectively; under 0.5 mM phosphorus level culture conditions, the inorganic phosphorus content in the leaves of wild-type plants ZH11, oshak25-1 mutant plants, and oshak25-2 mutant plants were 1.77 mg / g, 3.03 mg / g, and 2.25 mg / g fresh weight, respectively; and under 0.1 mM phosphorus level culture conditions, the inorganic phosphorus content in the leaves of wild-type plants ZH11, oshak25-1 mutant plants, and oshak25-2 mutant plants were 0.08 mg / g, 0.25 mg / g, and 0.20 mg / g fresh weight, respectively. The experimental results showed that the oshak25-1 mutant plant and the oshak25-2 mutant plant were 2 times and 1.3 times higher than the wild-type plant ZH11, respectively, indicating that the oshak25-1 mutant plant and the oshak25-2 mutant plant have stronger phosphorus uptake capacity compared with the wild-type plant ZH11.

[0060] Example 3: Expression of phosphorus uptake-related genes in the roots and aboveground parts of rice oshak25 mutant plants

[0061] Wild-type plants ZH11, oshak25-1 mutant, and oshak25-2 mutant were cultured in Kimura nutrient solution containing 0.2 mM phosphorus. After two weeks, total RNA was extracted from the roots and aboveground parts of the wild-type plants ZH11, oshak25-1 mutant, and oshak25-2 mutant and reverse transcribed into cDNA. Real-time quantitative PCR was performed using cDNA as a template to detect the expression levels of phosphorus uptake-related genes such as OsSPX2, OsPT2, OsPT3, and OsPAP21b in different materials. OsActin3 was used as an internal control gene. The above experiment was repeated three times, and the average value was taken as the experimental result.

[0062] The specific primer sequences are as follows:

[0063] OsSPX2-qpcr-F: 5'-GGAGGTGAAAACGAGAATGG-3';

[0064] OsSPX2-qpcr-R: 5'-ACAGCAGGTGGGAAACAAAC-3';

[0065] OsPT2-qpcr-F: 5'-CACAAACTTCCTCGGTATGCT-3';

[0066] OsPT2-qpcr-R: 5'-GAAACCCCACAAATCCACAAC-3';

[0067] OsPT3-qpcr-F: 5'-CATGCTCATGACGCTGCT-3';

[0068] OsPT3-qpcr-R: 5'-GCGACGTTCTCCTTGGAC-3';

[0069] OsPAP21b-qpcr-F: 5'-CAATCGTGATATCGTGTGTTTCAGC-3';

[0070] OsPAP21b-qpcr-R: 5'-AGCGAAAGATGAGCCCATCAGTG-3';

[0071] OsActin3-qpcr-F: 5'-CCACTATGTTCCCTGGCATT-3';

[0072] OsActin3-qpcr-R: 5'-GTACTCAGCCTTGGCAATCC-3'.

[0073] like Figure 3 As shown, the relative expression levels of phosphorus absorption-related genes such as OsSPX2, OsPT2, OsPT3, and OsPAP21b in the aboveground parts and roots of wild-type plants ZH11, oshak25-1 mutant plants, and oshak25-2 mutant plants were analyzed. The results showed that the expression levels of OsSPX2 gene in the aboveground parts and roots of oshak25-1 mutant plants and oshak25-2 mutant plants were significantly lower than those in wild-type plant ZH11. Specifically, the expression level of OsSPX2 gene in the roots of oshak25-1 mutant plants and oshak25-2 mutant plants was 9.3 times and 5.8 times lower than that in wild-type plant ZH11, respectively; the expression level of OsSPX2 gene in the aboveground parts of oshak25-1 mutant plants and oshak25-2 mutant plants was 2.2 times and 1.7 times lower than that in wild-type plant ZH11, respectively. However, the expression levels of OsPT2, OsPT3, and OsPAP21b genes in the roots of the oshak25-1 and oshak25-2 mutant plants were significantly higher than those in the wild-type plant ZH11. Specifically, the expression level of OsPT2 gene in the roots of the oshak25-1 and oshak25-2 mutant plants was 2.3 times and 2.8 times higher than that in the wild-type plant ZH11, respectively; the expression level of OsPT3 gene in the roots of the oshak25-1 and oshak25-2 mutant plants was 2.0 times and 2.9 times higher than that in the wild-type plant ZH11, respectively; and the expression level of OsPAP21b gene in the roots of the oshak25-1 and oshak25-2 mutant plants was 0.8 times and 1.4 times higher than that in the wild-type plant ZH11, respectively. The OsSPX2 gene is a key negative regulator of phosphorus starvation signaling, while the core positive regulator of this negative signaling is the OsPHR2 gene. The OsPT2 and OsPT3 genes are high-affinity phosphorus transporters, both involved in phosphate uptake and transport. Both genes have a P1BS cis-acting element in their promoters and may be directly regulated by the OsPHR2 gene. The OsPAP21b gene encodes purple acid phosphatase and is transcribedly regulated by the OsPHR2 gene. These results suggest that the oshak25-1 and oshak25-2 mutant plants may promote phosphorus uptake in rice by downregulating the expression of the OsSPX2 gene and increasing the expression of phosphorus-related genes such as OsPT2, OsPT3, and OsPAP21b.

[0074] It is understood that the above embodiments only illustrate preferred embodiments of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can freely combine the above technical features without departing from the concept of the present invention, and can also make several modifications and improvements, all of which fall within the protection scope of the present invention. Therefore, all equivalent transformations and modifications made with respect to the scope of the claims of the present invention should fall within the scope of the claims of the present invention.

Claims

1. The application of the potassium transporter gene OsHAK25 in improving phosphorus uptake in rice, characterized in that, The nucleotide sequence of the OsHAK25 gene is shown in SEQ ID NO.

2. The OsHAK25 gene negatively regulates the phosphorus uptake capacity of rice.

2. The application according to claim 1, characterized in that, The CDS sequence of the OsHAK25 gene is shown in SEQ ID NO.

3.

3. The application according to claim 2, characterized in that, The amino acid sequence of the protein encoded by the CDS sequence of the OsHAK25 gene is shown in SEQ ID NO.

1.

4. The application according to claim 1, characterized in that, The OsHAK25 gene was knocked out to obtain the oshak25 mutant plant. The inorganic phosphorus content in the leaves of the oshak25 mutant plant was significantly higher than that of the wild type under phosphorus treatment.

5. The application according to claim 4, characterized in that, The expression level of the OsSPX2 gene in the oshak25 mutant plant was lower than that in the wild-type plant, and the expression level of the OsSPX2 gene was negatively correlated with the phosphorus uptake capacity of rice.

6. The application according to claim 5, characterized in that, The expression level of the OsSPX2 gene was reduced to increase the expression levels of the OsPT2, OsPT3, and OsPAP21b genes, which are used to positively regulate the phosphorus uptake capacity of rice.

7. A breeding method for rice with high phosphorus absorption function, characterized in that, The method of knocking out the OsHAK25 gene in rice as described in any one of claims 1-6 comprises: Design target primers for sgRNA, wherein the target primers for sgRNA include OsHAK25-U3F and OsHAK25-U3R; A knockout vector was constructed, wherein the knockout vector was OsHAK25 CRISPR / Cas9; The knockout vector was transferred into an Agrobacterium-mediated transformation, and the Agrobacterium-mediated transformation was used to infect rice callus tissue to obtain transgenic rice plants T0. The oshak25 mutant plant was screened from the transgenic rice plant T0.

8. The breeding method according to claim 7, characterized in that, The target primer sequence for the sgRNA is as follows: OsHAK25-U3F: 5'-ggcaGTGAGAGGGCTCGGTACACA-3'; OsHAK25-U3R: 5'-aaacTGTGTACCGAGCCCTCTCAC-3'.

9. The breeding method according to claim 7, characterized in that, The construction of the knockout vector includes: The target primers of the sgRNA were annealed into double strands to obtain the target fragment; The pYLCRISPR-Cas9-gxy plasmid was digested with BsaI restriction enzyme to obtain the vector pYLCRISPR / Cas9-L; The target fragment was ligated to the vector pYLCRISPR / Cas9-L using T4 ligase to obtain the knockout vector OsHAK25 CRISPR / Cas9.

10. The breeding method according to claim 7, characterized in that, The process of screening oshak25 mutant plants from transgenic rice plants T0 includes: Primers were designed to target the genomic DNA of transgenic rice plant T0, and target DNA fragments were obtained. The target DNA fragment was amplified using PCR technology to obtain the amplified target DNA fragment. The amplified target DNA fragments were sequenced to screen out homozygous oshak25 mutant plants that had Cas9 removed.

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