Application of rice low-potassium adaptability related gene OsWRKY19

Abstract

The invention belongs to the field of plant genetic engineering, and particularly relates to a rice low-potassium adaptability related gene OsWRKY19 and application thereof. According to the invention, it is found for the first time that OsWRKY19 is a typical low-potassium response type transcription factor, and the growth and potassium accumulation ability of plants under a low-potassium condition is enhanced by positively regulating and controlling the expression level of the OsWRKY19 gene. The regulation mode comprises gene editing, promoter modification or exogenous overexpression, and the OsWRKY19 or functional equivalents thereof can be used as a molecular marker or a breeding target and is used for screening low-potassium efficient utilization type rice genotypes and cultivating new potassium efficient utilization varieties. The invention discloses a new function of the OsWRKY19 gene in potassium absorption and transportation, provides new gene resources and molecular targets for rice molecular design and breeding, and has important application values for reducing potassium fertilizer investment, improving crop nutrient utilization efficiency and realizing green and efficient agriculture.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering, specifically to the application of OsWRKY19 in the adaptation of rice to low potassium. Background Technology

[0002] Potassium is one of the essential macroelements for plant growth and development, playing a crucial role in osmotic regulation, enzyme activation, photosynthesis, ion balance, and signal transduction. Rice, as my country's most important food crop, has an extremely high demand for potassium. However, due to insufficient available potassium supply in the soil, low fertilizer utilization efficiency, and the environmental risks associated with excessive fertilization, rice exhibits poor adaptability under low potassium stress, which is a significant factor limiting yield and quality improvement.

[0003] In agricultural production, potash fertilizer is the third largest fertilizer after nitrogen and phosphate fertilizers, and my country is the world's largest consumer of potash fertilizer. For a long time, due to the limited availability of potash resources in my country, the country has been highly dependent on imports for potash fertilizer. Domestic potash fertilizer self-sufficiency is less than half, import dependence exceeds 50%, and annual imports often reach tens of millions of tons. At the same time, geopolitical fluctuations and commodity price volatility significantly impact fertilizer prices and affect the stability of agricultural production. Therefore, improving crop adaptability to low-potassium environments and cultivating new potash-efficient rice varieties are crucial to alleviating potash fertilizer dependence and reducing production costs.

[0004] Transcription factors play a central regulatory role in plant responses to nutrient stress. The WRKY transcription factor family is a plant-specific gene family known to participate in various stress responses and nutrient signal regulation. However, the function of WRKY family members in the low potassium response in rice remains unstudied.

[0005] The sequence of the gene OsWRKY19 has been published in the National Rice Data Center database (https: / / www.ricedata.cn / ). OsWRKY19 is a transcription factor unique to rice, currently known to enhance rice resistance to rice blast and bacterial blight. In addition, OsWRKY19 can directly bind to the promoter of the aroma-related gene OsBADH2 and inhibit its activity, thereby positively regulating the synthesis of 2-acetyl-1-pyrrolline (2-AP), a core substance of rice aroma.

[0006] The only known transcription factor involved in low potassium adaptation in rice is OsNAC25 of the NAC family, but it does not belong to the same gene family as OsWRKY19. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide an application of OsWRKY19 in the low potassium adaptation of rice.

[0008] To address the aforementioned technical problems, this invention provides the use of the gene OsWRKY19 for regulating low-potassium adaptation in rice. The nucleotide sequence of the gene OsWRKY19 is shown in SEQ ID NO.1, and the encoded amino acid sequence is shown in SEQ ID NO.2; or a nucleic acid molecule that has at least 90% sequence identity with the sequence and has low-potassium response regulation function.

[0009] That is, by positively regulating the expression level of the OsWRKY19 gene, the regulation methods include gene editing, promoter modification or exogenous overexpression, thereby enhancing the plant's growth and potassium accumulation capacity under low potassium conditions.

[0010] An improvement to the use of the gene OsWRKY19 of this invention: to improve the adaptability of rice to low potassium stress and the efficiency of potassium fertilizer utilization.

[0011] As a further improvement to the use of the gene OsWRKY19 of the present invention: regulating the plant's growth and potassium accumulation capacity under low potassium conditions.

[0012] Further improvements to the use of the gene OsWRKY19 of this invention include: reducing potassium fertilizer application, improving rice yield stability, and achieving green and efficient agricultural production.

[0013] As a further improvement to the use of the gene OsWRKY19 of the present invention: knocking out OsWRKY19 reduces the plant's growth and potassium accumulation capacity under low potassium conditions.

[0014] As a further improvement to the use of the gene OsWRKY19 of the present invention: overexpression of OsWRKY19 can enhance the growth and adaptability of plants under low potassium conditions.

[0015] As a further improvement to the use of the gene OsWRKY19 of the present invention: the gene OsWRKY19 is used as a molecular marker or breeding target to screen for low potassium-efficient rice genotypes or to breed new potassium-efficient rice varieties, or to breed potassium-efficient varieties by increasing the expression level of OsWRKY19 in rice to adapt to low potassium environments.

[0016] As a further improvement to the use of the gene OsWRKY19 of the present invention: the OsWRKY19 gene is significantly induced to express in rice roots under low potassium stress conditions, and reaches its expression peak on days 3 to 7 of treatment, which is a typical low potassium responsive transcription factor.

[0017] The purpose of this invention is to provide an application of the rice low-potassium adaptation regulatory gene OsWRKY19 and its encoded protein in improving rice's adaptation to low-potassium stress and efficient absorption and utilization of potassium nutrients. This invention is the first to discover that the OsWRKY19 gene can respond to low-potassium stress signals and plays a key role in regulating potassium absorption, translocation, and plant architecture development in rice, revealing its novel function and application value in the formation of low-potassium adaptation in rice.

[0018] The OsWRKY19 gene provided by this invention has the genomic nucleotide sequence shown in SEQ ID NO.1 and the amino acid sequence of its coding region shown in SEQ ID NO.2. This gene is derived from the japonica rice variety “Zhonghua 11” (ZH11). Under low potassium stress, this gene shows significant inducible expression in the roots, reaching a peak on days 3-7 of treatment, and is a typical low potassium-responsive transcription factor.

[0019] For those skilled in the art, the clone, expression vector, and transformed plants of this gene can be obtained using conventional molecular biology methods based on the described sequence. Nucleotide sequences (including mutants, alleles, or derivatives formed by the addition, substitution, insertion, or deletion of one or more bases) that have more than 90% homology with the sequence shown in SEQ ID NO.1 or SEQ ID NO.2 and maintain the same biological function are all within the scope of protection of this invention. Similarly, conserved variants, bioactive fragments, or derivatives that have more than 95% homology with the amino acid sequence shown in SEQ ID NO.2 and exhibit low potassium response function are also within the scope of protection of this invention.

[0020] This invention, through potassium deficiency treatment analysis, revealed that the expression level of OsWRKY19 in rice roots was significantly upregulated, reaching its highest level between days 3 and 7 of treatment, exhibiting typical low-potassium induced expression characteristics. This result indicates that OsWRKY19 is a typical low-potassium responsive gene, involved in the physiological response of rice to low-potassium stress.

[0021] To verify its function, this invention utilized the CRISPR-Cas9 system to perform site-specific knockout of OsWRKY19 in rice ZH11, obtaining homozygous mutants wrky19-1 and wrky19-2. Both mutants exhibited insertion or deletion at the target site, leading to frameshift or premature termination, forming loss-of-function homozygous mutant lines. Simultaneously, a 35S promoter-driven OsWRKY19 overexpression vector was constructed, and two stable overexpression lines (35s-WRKY19-1 and 35s-WRKY19-2) were obtained. Under greenhouse hydroponic conditions, the mutant plants exhibited slowed growth, abnormal leaf development, reduced tiller number, and significantly lower overall biomass than the wild type.

[0022] The results of culturing the mutant for 21 days at different potassium ion concentrations (1000 μM, 500 μM, 100 μM, 10 μM) showed that the mutant was sensitive to low potassium stress. Its aboveground and root biomass was significantly lower than that of the wild type, and the potassium content of the aboveground tissues was also significantly lower than that of the wild type. This indicates that the OsWRKY19 deletion reduced the ability to absorb and accumulate potassium, thus weakening the plant's adaptability to low potassium.

[0023] Furthermore, field trials with different potassium application treatments showed that the mutants wrky19-1 and wrky19-2 were weaker than the wild type in terms of tiller number, plant height, and overall growth under low, medium, and high potassium treatments. In contrast, the overexpression lines 35s-WRKY19-1 and 35s-WRKY19-2 exhibited higher effective tiller numbers and better growth vigor. Notably, as the potassium fertilizer application rate decreased, the growth difference between the mutants and the wild type further widened, and the growth advantage of the overexpression lines became more pronounced, further validating the key role of OsWRKY19 in improving the low potassium tolerance of rice.

[0024] In summary, this invention reveals for the first time the role of OsWRKY19 in the low-potassium response and potassium uptake regulation in rice, clarifying its key functions in plant growth and potassium homeostasis maintenance. The OsWRKY19 gene or its functional equivalents can also serve as molecular markers for screening and identifying low-potassium, high-efficiency-utilization rice genotypes, thus aiding in the breeding of new rice varieties with high potassium utilization efficiency. This invention also provides a molecular modification approach based on the OsWRKY19 gene to improve the efficiency of potassium uptake and utilization in rice. The method can regulate the expression level of OsWRKY19 through molecular biology techniques such as gene editing, promoter modification, or transgenics to improve rice's adaptability to low-potassium stress and its potassium utilization efficiency.

[0025] This invention is the first to discover that OsWRKY19 is a typical low-potassium-responsive transcription factor. Functional analysis shows that OsWRKY19 regulates potassium absorption, translocation, and plant architecture in rice, playing a crucial role in maintaining potassium homeostasis and promoting growth. After constructing OsWRKY19 loss-of-function mutants using CRISPR-Cas9 gene editing technology, the mutants exhibited phenotypes such as slowed growth, reduced tillering, and significantly decreased potassium content in the aboveground parts under low-potassium culture conditions, indicating that this gene plays an important regulatory role in the formation of low-potassium adaptation in rice. Field experiments showed that increasing the expression level of OsWRKY19 can significantly enhance the low-potassium adaptation of rice. OsWRKY19 or its functional equivalents can be used as molecular markers or breeding targets for screening low-potassium-efficient rice genotypes and breeding new potassium-efficient varieties. This invention reveals a novel function of the OsWRKY19 gene in potassium absorption and transport, providing new gene resources and molecular targets for molecular design breeding of rice, and has important application value for reducing potassium fertilizer input, improving crop nutrient utilization efficiency, and realizing green and efficient agriculture.

[0026] This invention, through expression analysis and gene editing function verification, identified that the WRKY transcription factor OsWRKY19 responds significantly under low potassium conditions and participates in regulating potassium absorption in rice. Functional verification shows that it participates in regulating potassium absorption, translocation, and plant development in rice, and can help plants maintain good growth and biomass accumulation under low potassium stress, which is consistent with the typical physiological characteristics of low potassium response in plants.

[0027] Therefore, OsWRKY19, as a key regulator of low potassium response, has significant potential applications in molecular design breeding and efficient nutrient utilization improvement of rice. By utilizing this gene, novel rice varieties with high yield potential and efficient potassium utilization under low potassium conditions can be obtained, thereby reducing potassium fertilizer input, lowering production costs, and improving food security and ecological sustainability. Attached Figure Description

[0028] The specific embodiments of the present invention will be further described in detail below with reference to the accompanying drawings.

[0029] Figure 1 This is a schematic diagram illustrating the expression analysis of the potassium deficiency response of OsWRKY19.

[0030] Figure 1 middle:

[0031] A. Changes in the relative expression level of OsWRKY19 in the aboveground parts after potassium deficiency treatment for 1, 3, 5, 7, and 14 days;

[0032] B. Changes in the relative expression level of OsWRKY19 in roots after 1, 3, 5, 7, and 14 days of potassium deficiency treatment.

[0033] Figure 2 A schematic diagram of the CRISPR / Cas9 editing vector structure used to construct the wrky19 mutant.

[0034] Figure 3 A schematic diagram of the construction of the OsWRKY19 overexpression vector.

[0035] Figure 4 A schematic diagram of the OsWRKY19 gene structure and mutant types, and a growth phenotype diagram of the mutants in a greenhouse;

[0036] Figure 4 middle:

[0037] A. Schematic diagram of the OsWRKY19 gene structure and mutant allele sequence constructed using the CRISPR / Cas9 system;

[0038] B, Tiller number statistics of wild-type ZH11 and mutants (wrky19-1 and wrky19-2);

[0039] C. Phenotypic characteristics of whole plants and leaves of wild-type ZH11 and homozygous mutants (wrky19-1 and wrky19-2) after 4 months of hydroponic cultivation in a greenhouse; data in the figure are expressed as mean ± SD, n≥3, *: P<0.05, **: P<0.01).

[0040] Figure 5 Phenotypes of wild-type and mutant at different potassium concentrations.

[0041] Figure 6 Statistics on biomass and potassium ion content of wild-type and mutant strains after different potassium concentration treatments;

[0042] Figure 6 middle:

[0043] A. Phenotypes of wild-type ZH11 and mutants (wrky19-1 and wrky19-2) after 21 days of culture at different potassium concentrations;

[0044] B, Aboveground biomass statistics;

[0045] C, Root biomass statistics;

[0046] D, Potassium ion content in the aboveground parts; E, Potassium ion content in the roots. (Data in the figure are expressed as mean ± SD, n≥3, *: P<0.05, **: P<0.01).

[0047] Figure 7The field phenotypes of ZH11, wrky19-1, wrky19-2, 35s-WRKY19-1 and 35s-WRKY19-2 lines under different potassium supply levels were determined. MK (medium potassium), LK (low potassium), and NK (no potassium) treatments were set up in the field.

[0048] Figure 7 middle:

[0049] A represents the whole-plant phenotype of each strain at maturity under MK, LK, and NK conditions;

[0050] B represents the number of effective tillers at maturity for each line under different potassium supply conditions;

[0051] C represents the plant height of each strain under different potassium supply conditions. Detailed Implementation

[0052] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto:

[0053] Example 1: Expression analysis of OsWRKY19 under low potassium conditions (qRT-PCR)

[0054] (1) Materials and growth conditions:

[0055] The rice variety Zhonghua 11 (ZH11) was soaked in 70% ethanol for 1 min and the liquid was discarded; it was then disinfected with 2% sodium hypochlorite for 10 min; and washed with sterile water 5-6 times. It was then placed in the dark at 28-30℃ for 36-48 h to germinate. After the radicles showed white, it was transferred to seedling trays and then placed in a greenhouse with Hoagland nutrient solution for rice improvement for 7 days.

[0056] The rice nutrient solution formula is as follows (standard formula):

[0057]

[0058] (2) Potassium treatment schemes at different levels:

[0059] ZH11 seedlings with uniform growth after 7 days of growth in a greenhouse were selected and treated with culture solutions of different potassium ion concentrations (500 μM and 10 μM). Aboveground parts and roots were collected at 1, 3, 5, 7, and 14 days after treatment, washed with ultrapure water, dried, flash-frozen in liquid nitrogen, and stored at -80℃. Three independent biological replicates (≥5 plants per replicate) were established for each time point and treatment. The nutrient solution formulations for different potassium ion treatments were modified from conventional nutrient solution formulations (KH2PO4 was replaced with NaH2PO4·2H2O and K2SO4 to separate potassium from phosphorus), as described below:

[0060] Basic salts:

[0061]

[0062] Trace element solution:

[0063]

[0064] Iron salt solution:

[0065] Element Target concentration (mM) NaFe-EDTA 0.04

[0066] The nutrient solution was diluted at a ratio of 1:1000 and used to treat rice. Different potassium treatments were then established: 1000 μM, 500 μM, 100 μM, and 10 μM. Note: For strict plasma strength control, a trace amount of Na2SO4 can be used to compensate for sulfate in the treatment; however, this embodiment follows the procedure of "reducing only K2SO4".

[0067] (3) Total RNA extraction and quality control:

[0068] Total RNA was extracted using the TRIzol method. Genomic DNA was removed by adding DNase I (e.g., 1 U / μg RNA, 37 °C, 15–30 min). After quality assessment, the following grades were selected: A. 260 / A 280 In 1.9~2.1; A 260 / A 230 Samples with a diameter ≥2.0 were then observed using 1% agarose gel electrophoresis to ensure clear and undegraded 28S / 18S bands.

[0069] (4) qRT-PCR reaction:

[0070] After reverse transcription of the above RNA, quantitative primers for OsWRKY19 were used.

[0071] wrky19-qRT-F: GAGGGGCAGATCATGCTGG

[0072] wrky19-qRT-R:TTGGCCATCCCCTCCTCTT

[0073] Run the reaction according to the following reaction system and procedure.

[0074] Reaction system (20 μL):

[0075] 2× SYBR Green Master Mix 10 μL Upstream primer (10 μM) 0.4 μL Downstream primer (10 μM) 0.4 μL cDNA 1 μg <![CDATA[ddH2O]]> Add to 20 μL

[0076] Loop program (regular):

[0077] 95 °C for 3 min;

[0078] 40 cycles: 95 °C for 10 s, 60 °C for 30 s (fluorescence collection);

[0079] Melting curve: 65~95 °C, heating rate 0.5 °C / step, confirmed single peak.

[0080] Each sample was tested in three replicates.

[0081] (5) Data processing:

[0082] For data processing, the Ct value of the reference gene was used for standardization, and ΔCt = Ct(target) - Ct(reference) was calculated. Using day 0 as the calibration sample, ΔCt = ΔCt(sample) - ΔCt(calibration) was calculated. Relative expression levels were calculated at 2... - The ΔΔCt method was used for calculation. Normalization analysis was performed using the internal reference gene OsActin1. Three biological replicates were set up for each time point, and two were used. - ΔΔCt is expressed as mean ± standard error (SEM). In statistical analysis, differences between two groups were analyzed using Student's t-test, and differences among multiple groups were analyzed using one-way ANOVA with Tukey's post-hoc test. The significance level was set at P < 0.05. Results showed that OsWRKY19 did not exhibit a significant trend of induction or inhibition by potassium deficiency treatment in the aerial parts, but showed significant induction in the roots, peaking on days 3–7 of treatment, classifying it as a typical low-potassium-responsive transcription factor. Figure 1 ).

[0083] Example 2: Construction of OsWRKY19 knockout vector

[0084] (1) Target identification and primer design:

[0085] Based on the reference genome, the genomic sequence (SEQ ID NO.1) and protein sequence (SEQ ID NO.2) of OsWRKY19 were obtained; the location of the conserved WRKY domain was confirmed using SMART and NCBI-CDD; subsequently, specific knockout primers were designed using the online primer design website CRISPR-P 2.0. A dual-target knockout strategy was employed for the OsWRKY19 gene.

[0086] Target 1:

[0087] W19-U3-F:ggcATTCTTGGCGCCAAATACCCG

[0088] W19-U3-R:aaacCGGGTATTTGGCGCCAAGAA

[0089] Target 2:

[0090] W19-U6a-F: gccGCCTACTTCAGGTGCACTCAC

[0091] W19-U6a-R:aaacGTGAGTGCACCTGAAGTAGG

[0092] (2) Synthesize target site adapter primers:

[0093] Dissolve the synthesized adapter primers in a 10 μM stock solution. Take 10 μL each of the F-direction and R-direction adapter primer stock solutions and mix them with 80 μL of water to dilute the adapters to a concentration of 1 μM. After denaturation at 95 °C for 1 min, cool to room temperature to complete the annealing.

[0094] (3) Enzyme digestion of gRNA vector:

[0095] Take 1 μg of pYLgRNA-OsU3 or pYLgRNA-OsU6a plasmid, add ~10 μL of UEcoR31I (Thermo Scientific) to a 25 μL reaction system, digest at 37 ℃ for 20 min, then inactivate the enzyme at 70 ℃ for 5 min, and store at -20 ℃ for later use. The reaction system is as follows:

[0096] reagents Usage 10 x Fast Digest Buffer 2 μL PYLgRNA-OsU3 / OsU6 up to 1 μg Eco31I (10 U / μL) 1 μL <![CDATA[ddH2O]]> Add to 20 μL

[0097] (4) gRNA expression cassette ligation reaction:

[0098] The annealed target adapter was ligated to the corresponding vector of enzyme-digested OsU3-gRNA or OsU6a-gRNA. The reaction system is as follows: ligation was carried out at room temperature (20~28 ℃) for 10~15 min.

[0099] gRNA expression cassette ligation reaction system:

[0100] reagents Usage 10 x T4 DNA ligase buffer 1 μL PYLgRNA-OsU3 / OsU6 (~20 ng) 0.5 μL Target adapter (final concentration 0.05 μM) 0.5 μL T4 DNA ligase (25~30 U) 0.5 μL <![CDATA[ddH2O]]> To bring the volume up to 10 μL

[0101] (5) Amplification of gRNA expression cassettes:

[0102] First round of amplification: 1 μL of the ligation product (obtained in step (4) above) was used as the PCR template. 0.2 μM each of UF (5-CTCCGTTTTACCTGTGGAATCG-3) and gRNA-R (5-CGGAGGAAAATTCCATCCAC-3) were used as adapter primers. The first round of amplification was performed using KOD-FX (TOYOBO) high-fidelity polymerase. The amplification program was: 95 ℃ for 2 min; 95 ℃ for 10 s, 60 ℃ for 15 s, 68 ℃ for 20 s, 25 cycles; and a final extension at 68 ℃ for 5 min. 4 μL was then examined by electrophoresis.

[0103] Second round of amplification: Take 1 μL of the product from the first round of PCR reaction, dilute it 10-fold with ddH2O, and use 1 μL as the template for the PCR reaction. Amplify T1-U3 using the gRNA expression cassette position-specific primers B1' (TTCAGAggtctcTctcgCACTGGAATCGGCAGCAAAGG) + B2 (AGCGTGggtctcGtcagGGTCCATCCACTCCAAGCTC), and amplify T2-U6a using B2' (TTCAGAggtctcTctgaCACTGGAATCGGCAGCAAAGG) + BL (AGCGTGggtctcGaccgGGTCCATCCACTCCAAGCTC). Perform the second round of PCR reaction. The amplification program is: 95 ℃ for 2 min; 95 ℃ for 10 s, 58 ℃ for 15 s, 68 ℃ for 20 s, 15 cycles; and a final extension at 68 ℃ for 5 min. Check the size of the PCR products by electrophoresis, and recover the PCR products by gel extraction.

[0104] (6) Ligating the gRNA expression cassette to the pYLCRISPR / Cas9-MH plasmid:

[0105] The PCR gel-recovered products were ligated with the pYLCRISPR / Cas9-MH plasmid using a ligation-digestion method. The ligation system is as follows: After mixing the reaction mixture, the mixture was incubated at 37 °C for 10 min. Then, 0.5 μL of 10 x NEB T4 DNA ligase buffer and ~35 U T4 DNA ligase were added for temperature-controlled cyclic ligation. The reaction process was: 37 °C for 2 min, 10 °C for 3 min, 20 °C for 5 min, for 15 cycles; and finally, 37 °C for 2 min.

[0106] pYLCRISPR / Cas9-MH support linkage reaction system:

[0107] reagents Usage gRNA expression cassette purified product (obtained in step (5)) 20-70 ng pYLCRISPR / Cas9-MH vector 60-80 ng 10 x EcoR31I endonuclease buffer 1.5 μL EcoR31I endonuclease (10 U) 1 μL <![CDATA[ddH2O]]> To bring the volume up to 15 μL

[0108] (7) Transformation:

[0109] The ligation product was transformed into *E. coli* DH5α using a chemical heat shock method. Positive clones were selected by colony PCR, and after plasmid verification by sequencing, it was transformed into *Agrobacterium* competent cells using a freeze-thaw method. *Agrobacterium*-mediated transformation of ZH11 callus was then used to construct the wrky19 mutant line. The recombinant vector was pYLCRISPR / Cas9-OsWRKY19. (Editing vector details omitted). Figure 2 As shown.

[0110] Example 3: Construction of OsWRKY19 overexpression vector

[0111] To obtain overexpression material of the OsWRKY19 gene, the full-length cDNA of OsWRKY19 was amplified by PCR and then directionally cloned into a plant expression vector derived from pCAMBIA1300. The vector contains the CaMV 35S promoter (a strong promoter) to drive the expression of the target gene and carries a GFP marker sequence for subcellular localization observation.

[0112] The primers used for PCR amplification are as follows:

[0113] Forward primer F: 5'- acgggggacgagctcggtaccATGGTGGAGCTCTGCGGC -3';

[0114] Reverse primer R: 5'- gcccttgctcaccatgtcgacCAGATTCTGAATCTCCGATTGGA-3';

[0115] The empty vector plasmid was double-digested with KpnI and Sal1, and then ligated with the amplified fragment to form the overexpression recombinant vector 35S:OsWRKY19-GFP. A schematic diagram of the recombinant vector is shown below. Figure 3 As shown. After transformation into E. coli DH5α, positive clones were screened, and the correctness of the inserted fragment was verified by sequencing. The correct 35S:OsWRKY19-GFP overexpression vector was verified to be suitable for Agrobacterium-mediated rice genetic transformation to obtain OsWRKY19 overexpression lines.

[0116] Example 4: Genetic transformation of rice (mature seed explants - Agrobacterium method)

[0117] Rice genetic transformation was performed using mature seed explants-Agrobacterium-mediated transformation to construct the OsWRKY19 gene-editing mutant. The specific implementation steps are as follows:

[0118] (1) Callus induction and subculture:

[0119] Mature seeds of rice variety ZH11 were selected as explants. After surface sterilization with 70% ethanol and sodium hypochlorite solution, they were inoculated into callus induction medium (MS + 2,4-D 2 mg / L + hydrolyzed casein 0.3 g / L + sucrose 30 g / L + agar 8 g / L, pH = 5.8) and cultured at 28 °C in the dark for 3-4 weeks to induce embryogenic callus. Dense, yellow, spherical embryogenic callus was selected and transferred to subculture medium. After subculturing for 1 week at the same temperature, healthy callus material was obtained.

[0120] (2) Agrobacterium infection and co-culture:

[0121] The transformed strain was EHA105, carrying the constructed pYLCRISPR / Cas9-OsWRKY19 editing vector and the overexpression vector 35S:OsWRKY19-GFP. Agrobacterium was inoculated into YEP liquid medium containing kanamycin and streptomycin and cultured at 28 °C with shaking at 250 rpm until OD200 was reached. 600 ≈ 0.6–0.8. Collect bacterial cells by centrifugation and resuspend in AAM solution containing 200 μM acetosyringone. Briefly co-culture the callus tissue in the bacterial culture (about 5–10 min), remove excess liquid, and transfer to co-culture medium (containing acetosyringone) for 2–3 days at 25 °C in the dark.

[0122] 3. Screening, culture, and differentiation:

[0123] After co-culture, the callus tissue was transferred to a culture medium containing antibiotics (hygromycin 50 mg / L). -1 and kanamycin 50 mg·L -1 Two rounds of selection were performed on the selective medium, each lasting about 2 weeks. The resistant callus obtained from the selection was transferred to differentiation medium (MS + NAA 0.5 mg / L + BAP 3 mg / L) and cultured at 25 °C under light for 30–40 days to induce budding.

[0124] 4. Rooting and Transplanting:

[0125] Plants that have differentiated shoots were transferred to rooting medium (1 / 2 MS + NAA 0.1 mg / L) and cultured until the root system was robust before transplanting. The agar was washed off before transplanting, and the plants were transferred to hydroponic containers or substrate and cultured at 28 °C. T0 generation plants were obtained after seedling establishment.

[0126] 5. Molecular detection and screening of homozygous mutants:

[0127] Genomic DNA was extracted from leaves of T0 generation plants. A 500–800 bp fragment was amplified using primers flanking the target region. The amplified product was recovered via gel electrophoresis and subjected to Sanger sequencing. Based on the sequencing results, two mutant types were selected as representatives, named wrky19-1 and wrky19-2. wrky19-1 and wrky19-2 exhibited base deletions / insertions at the target site, leading to frameshifts or premature termination, indicating loss-of-function alleles. Simultaneously, total RNA was extracted from the overexpression lines, reverse transcribed, and analyzed by qRT-PCR. The results showed that the transcription level of WRKY19 in the WRKY19-OE line was significantly higher than that in the wild type (NIP). OsActin1 was used as an internal reference gene for normalization analysis. Through the above detection and validation, the WRKY19 knockout line (wrky19-cr) and the overexpression line (WRKY19-OE) were finally obtained. Homozygous positive seedlings were propagated to the T2 generation for subsequent functional validation and phenotypic analysis. The nucleotide sequence of wrky19-1 is shown in SEQ ID NO:3, and the nucleotide sequence of wrky19-2 is shown in SEQ ID NO:4.

[0128] Example 5: Phenotypic and Functional Confirmation

[0129] (1) Observation of greenhouse hydroponics and plant type phenotype:

[0130] Homozygous wrky19-1 and wrky19-2 mutants, as well as wild-type ZH11, identified by PCR and sequencing, were selected as experimental materials and cultured hydroponically under greenhouse conditions. The greenhouse temperature was maintained at 28 ± 2 °C (daytime) / 22 ± 2 °C (nighttime), and the light intensity was approximately 500 μmol·m⁻². -2 ·s -1 The photoperiod was 14 h light / 10 h dark, and the relative humidity was maintained at 60%–70%. Modified Hoagland nutrient solution was used as the culture medium, and the solution was changed every 7 days. After 4 months of cultivation, the overall growth status and leaf morphology of the plants were observed. Representative plants were photographed, and plant height, number of tillers, and above-ground dry weight of a single plant were recorded. Tillers were counted manually, with 6 plants randomly selected from each material (n≥6) for replication, and the average value was calculated.

[0131] The results showed that the growth of wrky19-1 and wrky19-2 plants was significantly inhibited compared with the wild type ZH11, with abnormal leaf development, lighter color, and a significantly reduced number of tillers. Figure 4 The overall biomass of the mutant was significantly lower than that of the wild type, indicating that the absence of OsWRKY19 led to a decrease in plant vigor.

[0132] (2) Treatment with different potassium concentrations:

[0133] To further analyze the effects of the OsWRKY19 gene on potassium uptake and low-potassium adaptation, one-week-old rice seedlings were transplanted into hydroponic systems with different potassium concentrations and cultured for 21 days. The nutrient solution used followed the strategy described in Example 1. Except for K... + All components except concentration were kept consistent. Four treatment groups were set up as follows: 1000 μM K + 500 μM K + 100 μM K + 10 μM K + During cultivation, the solution pH was maintained at approximately 5.8, and the solution was changed every 3 days. After treatment, the whole plant phenotype and the dry weight of the aboveground parts and roots were recorded. The results showed that under different potassium treatment conditions, the biomass of all roots in the aboveground parts of the mutant lines wrky19-1 and wrky19-2 were lower than those of the wild type, and the potassium content in the aboveground parts was significantly lower than that of the wild type. Figure 6 The potassium ion content in the roots was not different from that in the wild type.

[0134] Example 6: Determination of potassium content in tissues

[0135] To determine the potassium ion content in plant tissues, the processed samples were dried, digested, and analyzed by ICP-MS (Inductively Coupled Plasma-Mass Spectrometry). The specific steps are as follows:

[0136] (1) Sample preparation:

[0137] The harvested aboveground and root samples were dried at 65 °C for 72 hours until constant weight, then ground into powder, and 0.2~0.3g was weighed and placed in a polytetrafluoroethylene digestion tube.

[0138] (2) Digestion with acid:

[0139] Add 5 mL of concentrated nitric acid (HNO3) + 1 mL of hydrogen peroxide (H2O2) to each sample.

[0140] (3) Microwave digestion conditions:

[0141] The CEM MARS 6 microwave digestion system was used.

[0142] The setup procedure is as follows:

[0143] step warming phase Duration Temperature (°C) Power (W) ① 8 min 5 min 160 Automatic adjustment ② 5 min 20 min 185 Automatic adjustment

[0144] During digestion, keep the exhaust and cooling systems running to ensure stable chamber pressure. The entire process takes approximately 40 minutes.

[0145] (4) Post-digestion treatment:

[0146] After digestion, cool to room temperature, open the lid, and evaporate to dryness on a 160 °C heating block.

[0147] Then, dilute to 50 mL with Milli-Q pure water, mix well, and let stand to clarify.

[0148] (5) ICP-MS determination:

[0149] Potassium content was determined using a PerkinElmer Optima 8000 ICP-OES analyzer. The analytical wavelength was 766.49 nm, and the standard solution (0–10 mg·L⁻¹) was used. -1 K + Plot calibration curves, with 3 biological replicates for each sample.

[0150] (6) Results Analysis:

[0151] The data obtained showed that under all four potassium treatment culture conditions, the biomass in the aboveground parts and roots of the mutant was significantly reduced. At the same time, the potassium content in the aboveground parts of the mutant samples was significantly reduced, while the potassium ion content in the roots was consistent with that of the wild type, indicating that the OsWRKY19 deletion weakened the potassium absorption and transport capacity.

[0152] The above experimental results indicate that OsWRKY19 exhibits significant inducible expression characteristics under low potassium stress, suggesting its important role in the perception and response of rice to low potassium signals. Functional validation results further demonstrate that OsWRKY19 influences plant nutrient homeostasis and growth status by regulating potassium absorption and accumulation. Regulating the expression level of this gene can significantly improve the growth performance of rice under low potassium conditions, maintain high biomass and potassium nutrient use efficiency, thereby enhancing the plant's low potassium adaptability.

[0153] Example 7: Field phenotype of OsWRKY19 transgenic material

[0154] To verify the adaptability of the OsWRKY19 gene to rice under low potassium conditions, a field experiment was conducted in nutrient gradient fields in Changxing, Zhejiang Province, using wild-type ZH11, homozygous mutants (wrky19-1, wrky19-2), and stably expressed overexpression lines (35s-WRKY19-1, 35s-WRKY19-2). Three potassium levels were set in the field: MK (medium potassium): 15 kg potassium chloride / mu; LK (low potassium): 7.5 kg potassium chloride / mu; NK (no potassium): 0 kg potassium chloride / mu. Other nutrients (nitrogen, phosphorus) and field management practices remained consistent. Each line was planted with 30 plants (6x5) in each nutrient gradient field, and plant height and effective tiller number were recorded at maturity. The field experiment showed that the wrky19 mutation reduced the low potassium adaptability of rice, manifested as a decrease in plant height and tiller number, especially in the low potassium and no potassium experimental fields. Conversely, overexpression of WRKY19 enhanced the low-potassium adaptability of rice, manifested as an increase in tiller number, and also a significant increase in plant height in both low-potassium and no-potassium experimental fields. Figure 7 ).

[0155] Finally, it should be noted that the above examples are merely some specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments and many variations are possible. All variations that can be directly derived or conceived by those skilled in the art from the disclosure of the present invention should be considered within the scope of protection of the present invention.

Claims

1. The use of gene OsWRKY19, characterized in that: To regulate the low potassium adaptation of rice, the nucleotide sequence of the gene OsWRKY19 is shown in SEQ ID NO.1, and the encoded amino acid sequence is shown in SEQ ID NO.2; or a nucleic acid molecule that has at least 90% sequence identity with the sequence and has the function of regulating low potassium response.

2. The use of the gene OsWRKY19 according to claim 1, characterized in that: Improve rice's adaptability to low potassium stress and potassium fertilizer utilization efficiency.

3. The use of the gene OsWRKY19 according to claim 2, characterized in that: Regulates plant growth and potassium accumulation under low potassium conditions.

4. The use of the gene OsWRKY19 according to claim 3, characterized in that: Reduce potassium fertilizer application, improve rice yield stability, and achieve green and efficient agricultural production.

5. The use of the gene OsWRKY19 according to any one of claims 1 to 4, characterized in that: Knocking out OsWRKY19 reduces the plant's growth and potassium accumulation capacity under low potassium conditions.

6. The use of the gene OsWRKY19 according to any one of claims 1 to 4, characterized in that: Overexpression of OsWRKY19 can enhance the plant's growth and adaptability under low potassium conditions.

7. The use of the gene OsWRKY19 according to any one of claims 1 to 4, characterized in that: The OsWRKY19 gene can be used as a molecular marker or breeding target to screen for low-potassium, high-efficiency rice genotypes or to breed new rice varieties with high potassium efficiency. Alternatively, by increasing the expression level of OsWRKY19 in rice to adapt to low-potassium environments, varieties with high potassium efficiency can be bred.

8. The use of the gene OsWRKY19 according to any one of claims 1 to 4, characterized in that: The OsWRKY19 gene was significantly induced in rice roots under low potassium stress, reaching its peak expression on days 3-7 of treatment, and is a typical low potassium-responsive transcription factor.

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