Application of OsZIP13 gene

Abstract

The invention provides application of an OsZIP13 gene. The OsZIP13 gene is used for regulating and controlling saline-alkali stress tolerance of rice plants. The OsZIP13 gene in the rice plant is over-expressed and is used for enhancing the saline-alkali stress tolerance of the rice plant; or / and the OsZIP13 gene in the rice plant is subjected to gene editing and is used for reducing the saline-alkali stress tolerance of the rice plant; the nucleotide sequence of the coding region of the OsZIP13 gene is as shown in SEQ ID No: 1; the amino acid sequence of the coding region of the OsZIP13 gene is as shown in SEQ ID No: 2. The gene is excessively expressed in rice, the tolerance of rice to saline-alkali stress in the germination stage and the seedling stage can be enhanced, a mutant strain is more sensitive to saline-alkali stress compared with wild rice, and the discovery of the gene provides an important gene resource and a theoretical basis for cultivation of saline-alkali tolerant plants; wide application prospects are realized.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the application of the OsZIP13 gene. Background Technology

[0002] Soil salinization refers to an environmental geological phenomenon caused by the redistribution, excessive accumulation, and diffusion of soluble salts in the soil under unfavorable natural conditions such as climate, hydrology, topography, and soil texture. It not only causes farmland degradation and desertification, disrupting ecosystems, but also severely restricts agricultural production and economic development. Iron is an essential micronutrient for plant growth and development, playing a crucial role in plant respiration, photosynthesis, protein and nucleic acid synthesis, and enzymatic reactions. Iron deficiency leads to inhibited chlorophyll synthesis, manifesting as yellowing of new leaves, and in severe cases, affecting crop yield and quality. Studies have shown that saline-alkali soils affect the absorption and translocation of iron ions in rice, thus impacting rice growth, development, yield, and quality. Therefore, how to cultivate new rice varieties adapted to saline-alkali soils by improving the rice's ability to absorb and translocate iron ions is a major problem urgently needing to be solved in agricultural production.

[0003] The ZIP family of rice genes are mainly involved in the transport of zinc and iron ions, and some family genes have been found to participate in the response to salt and alkali stress. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to address the shortcomings of the prior art by providing an application of the OsZIP13 gene. Overexpression of this gene in rice can enhance the rice’s tolerance to salt and alkali stress during the germination and seedling stages, providing important gene resources and theoretical basis for cultivating salt-tolerant plants, and has broad application prospects.

[0005] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is: an application of the OsZIP13 gene, wherein the OsZIP13 gene is used to regulate the salt and alkali stress tolerance of rice plants; Overexpression of the OsZIP13 gene in rice plants was used to enhance the salt and alkali stress tolerance of the rice plants. Or / and gene editing of the OsZIP13 gene in rice plants to reduce the salt and alkali stress tolerance of said rice plants; The nucleotide sequence of the coding region of the OsZIP13 gene is shown in SEQ ID No:1; the amino acid sequence of the coding region of the OsZIP13 gene is shown in SEQ ID No:2.

[0006] Preferably, when the OsZIP13 gene in rice plants is overexpressed, the root length of rice plants under salt-alkali stress during the germination period is higher than that of wild-type rice plants. Under salt-alkali stress, the fresh weight, plant height, root length, and chlorophyll content of rice seedlings were all higher than those of wild-type rice plants, while the MDA content was lower than that of wild-type rice plants.

[0007] Preferably, when the OsZIP13 gene in rice plants is edited, oszip13 mutant rice plants are obtained. Under salt-alkali stress, the plant height and root length of the oszip13 mutant rice plants are both lower than those of wild-type rice plants.

[0008] Preferably, an overexpression vector of the OsZIP13 gene is constructed for the cultivation of transgenic rice plants with high tolerance to salt and alkali stress.

[0009] Compared with the prior art, the present invention has the following advantages: This invention constructs a plant expression vector for the OsZIP13 gene through homologous cloning, overexpresses the OsZIP13 gene in rice to obtain transgenic plants, and simultaneously constructs a CRISPR / Cas9 mutant, obtaining the rice zip13 mutant through gene editing. The salt-alkali tolerance function of the OsZIP13 gene is verified in detail using the overexpression lines and the mutant lines. The experiments of this invention demonstrate that the OsZIP13 gene has strong alkali tolerance. Overexpression of this gene in rice significantly enhances the rice's tolerance to salt-alkali stress during germination and seedling stages, and the mutant lines are more sensitive to salt-alkali stress than wild-type rice. The discovery of this gene provides important genetic resources and theoretical basis for breeding salt-alkali tolerant plants and has broad application prospects.

[0010] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments. Attached Figure Description

[0011] Figure 1 This is a clone diagram of the rice OsZIP13 gene in Example 1 of the present invention.

[0012] Figure 2 This is a graph showing the expression level analysis of the OsZIP13 gene in different tissues and organs of rice in Example 1 of the present invention.

[0013] Figure 3 This is a diagram illustrating the expression pattern of the rice OsZIP13 gene under salt-alkali stress in Example 1 of the present invention.

[0014] Figure 4 This is a diagram showing the expression pattern of the rice OsZIP13 gene under iron deficiency stress in Example 1 of the present invention.

[0015] Figure 5 This is a diagram showing the expression pattern analysis of the rice OsZIP13 gene under zinc deficiency stress in Example 1 of the present invention.

[0016] Figure 6 This is a diagram showing the expression pattern of the rice OsZIP13 gene under cadmium stress in Example 1 of the present invention.

[0017] Figure 7 This is an identification diagram of the rice OsZIP13 gene mutant plant in Example 2 of the present invention; wherein, A is a schematic diagram of the oszip13 mutant target site; B is the detection of the oszip13 mutant target effect.

[0018] Figure 8 This is a phenotypic analysis diagram of rice oszip13 gene mutant plants under salt-alkali stress during germination in Example 2 of the present invention; wherein, A is the phenotypic analysis under salt-alkali stress; B is the measurement of shoot length; and C is the measurement of root length.

[0019] Figure 9 This is a detection diagram of the resistance gene in rice plants overexpressing the OsZIP13 gene in Example 2 of the present invention.

[0020] Figure 10 This is a graph showing the expression level of the OsZIP13 gene in the transgenic plant in Example 2 of the present invention.

[0021] Figure 11 This is a phenotypic analysis diagram of rice plants overexpressing the OsZIP13 gene under alkali stress during germination in Example 2 of the present invention; A is the phenotypic analysis during germination; B is the measurement of shoot length; C is the measurement of root length.

[0022] Figure 12 This is a phenotypic analysis diagram of rice seedlings under alkaline stress during the OsZIP13 gene overexpression stage in Example 2 of the present invention; A is the phenotypic analysis of the seedling stage; B is the statistics of fresh weight; C is the measurement of plant height; D is the measurement of root length.

[0023] Figure 13 This is a graph showing the chlorophyll content detection of rice plants overexpressing the OsZIP13 gene in Example 2 of the present invention.

[0024] Figure 14 This is a graph showing the malondialdehyde (MDA) content detection in rice plants overexpressing the OsZIP13 gene in Example 2 of the present invention. Detailed Implementation

[0025] Example 1

[0026] This example is an analysis of the expression pattern of the rice OsZIP13 gene under stress.

[0027] 1. Cloning of the rice OsZIP13 gene: (1) Treatment of plant materials: Select plump seeds of the Nipponbare rice variety. First, soak the seeds in water for 24 hours, then rinse them four times and place them on moist filter paper. Incubate them in the dark at 28℃ for two days to promote rooting and germination. When the roots grow to about 2cm, transfer them to 96-well plates and place them in Kimura B nutrient solution (pH 5.8, formula as follows: 365μM (NH4)2SO4, 182μM KH2PO4, 183μM KNO3, 86μM K2SO4, 543μM MgSO4, 366μM Ca(NO3)2, 46.2μM H3BO3, 9.19μM MnCl2, 0.5μM H2MoO4, 0.77μM ZnSO4, 0.32μM CuSO4, 20μM Na2EDTA and 20μM The roots were immersed in the culture medium (FeSO4, all reagents were analytical grade reagents from Sinopharm brand) in a culture box, and then placed in an artificial climate chamber for cultivation. After 7 days, when the seedlings had grown, the root tissue was taken and quickly frozen in liquid nitrogen, and then stored at -80℃ for later use.

[0028] (2) RNA extraction: Total RNA was extracted from the rice roots using the Trizol method (TIANGEN).

[0029] (3) Obtaining cDNA: Using the total RNA as a template, cDNA was obtained by reverse transcription using a reverse transcription kit (TIANGEN).

[0030] (4) PCR amplification: Using the cDNA of the above-mentioned Nipponbare rice variety as a template, PCR amplification was performed using Phanta high-fidelity DNA polymerase (Novizan, catalog number P516) to obtain the PCR amplification product. The PCR amplification reaction system was as follows: 2×PhantaMax Buffer 25µL, dNTP Mix (10mM) 1µL, cDNA 1µL, upstream primer 2µL, downstream primer 2µL, PhantaDNA Polymerase 1µL, and ddH2O to a final volume of 50µL. The PCR amplification reaction program was as follows: 95℃ for 3 min; 95℃ for 15 s, 60℃ for 15 s, 35 cycles; 72℃ for 40 s; 72℃ for 5 min; and the reaction was terminated at 4℃. The nucleotide sequence of the OsZIP13 gene coding region is shown in SEQ ID No:1, and the amino acid sequence of the OsZIP13 gene coding region is shown in SEQ ID No:2.

[0031] The nucleotide sequence of the upstream primer is shown in SEQ ID No:3, and the nucleotide sequence of the downstream primer is shown in SEQ ID No:4.

[0032] The PCR amplification products were detected by 1.2% agarose gel electrophoresis. Figure 1 As shown, a band with a molecular weight of approximately 1.2 kb was obtained, which is consistent with the expected band size. The gel block containing the target band was cut off and sent to a sequencing company for sequencing.

[0033] The sequencing results were compared with sequences in the rice genome database, and the results showed that the PCR amplification product was indeed the OsZIP13 gene, with a length of 1173 bp, containing the complete ORF region.

[0034] 2. Relative expression levels of the rice OsZIP13 gene in different tissues and organs of rice: (1) Treatment of plant materials: Select plump seeds of the Japanese rice variety Nipponbare. First, soak the seeds in water for 24 hours, then rinse them four times and place them on moist filter paper. Incubate them in the dark at 28℃ for two days to promote rooting and germination. When the roots grow to about 2cm, transfer them to seedling pots containing Kimura B nutrient solution and place them in an artificial climate chamber for cultivation. When the seedlings reach 2 weeks of age, take rice root, stem, and leaf tissue materials and freeze them rapidly in liquid nitrogen, storing them at -80℃ for later use. Continue to cultivate the remaining seedlings until the rice heading stage. Take the newly emerged rice panicles and freeze them rapidly in liquid nitrogen, storing them at -80℃ for later use. Continue to cultivate until the late grain-filling stage, then take rice seeds and freeze them rapidly in liquid nitrogen, storing them at -80℃ for later use.

[0035] (2) Extraction of total RNA and acquisition of cDNA: Total RNA was extracted from the different tissue samples obtained above using Trizol reagent (TIANGEN); the total RNA was then used as a template to obtain the first strand of cDNA through reverse transcription using the FastKing RT Kit (TIANGEN).

[0036] (3) Real-time quantitative PCR: Using the first strand of the cDNA as a template, the expression level of the OsZIP13 gene in different tissues of rice was detected using the OsZIP13 gene characteristic primers OsZIP13-qRTS and OsZIP13-qRTAS with the real-time quantitative PCR reagent SuperReal PreMix Plus (TIANGEN). The reaction system for real-time quantitative PCR was as follows: 5 µL of 2×SuperRealPreMix Plus, 1 µL of cDNA, 0.3 µL of upstream primer, 0.3 µL of downstream primer, and RNase-free ddH2O to a final volume of 10 µL. The PCR amplification program was as follows: 95℃ for 15 min; 95℃ for 10 s, 60℃ for 20 s, 40 cycles; 72℃ for 30 s. The nucleotide sequence of the upstream primer OsZIP13-qRTS is shown in SEQ ID No:5, and the nucleotide sequence of the downstream primer OsZIP13-qRTAS is shown in SEQ ID No:6.

[0037] Real-time quantitative PCR used the comparative CT method (ΔΔCT) to calculate gene expression levels, with the rice Ubi5 gene as the internal reference gene. Differences in target gene expression were represented by the fold increase in expression level in each tissue sample relative to the expression level in the rice root sample. Each sample included three biological replicates and three technical replicates. Data were taken as the average of the three biological replicates; if one value showed a large deviation, the average of the two data points was used. Raw data were standardized, and the standardized data were analyzed for significance using a T-test. The relative expression level was calculated as follows: 2 -ΔΔCT =2 - ( ΔCT处理 - ΔCT对照 )=2 -[(CT处理目的基因-CT处理内参基因)-(CT对照目的基因-CT对照内参基因)] The nucleotide sequence of the upstream primer of the internal reference gene is shown in SEQ ID No:7, and the nucleotide sequence of the downstream primer is shown in SEQ ID No:8.

[0038] The results are as follows Figure 2 As shown, the OsZIP13 gene is expressed in all tissues of rice plants, and its expression level in the aboveground parts is significantly higher than that in the underground parts, especially in the leaves, where the expression level is the highest, about 17 times that in the roots. This indicates that the OsZIP13 gene is an important gene in the growth and development of rice and mainly functions in the aboveground parts.

[0039] 3. Analysis of the expression pattern of the OsZIP13 gene under salt-alkali stress: (1) Treatment of plant materials: Select plump seeds of the Japanese rice variety Nipponbare. First, soak the seeds in water for 24 hours, then rinse them four times and place them on moist filter paper. Incubate them in the dark at 28°C for two days to promote rooting and germination. When the roots grow to about 2 cm, transfer them to a 96-well plate and place them in a container filled with Kimura B nutrient solution, immersing the roots in the nutrient solution. Place them in an artificial climate chamber for cultivation. When the rice seedlings reach 2 weeks of age, transfer them to normal Kimura B nutrient solution and Kimura B nutrient solution containing 50 mM NaHCO3 (pH 8.3) for alkaline stress treatment. After 12 hours of treatment, select three rice seedlings with the same growth rate, and cut their root and leaf tissues as test samples. Quickly freeze them in liquid nitrogen and then store them at -80°C for later use.

[0040] (2) Extraction of total RNA and acquisition of cDNA: Total RNA was extracted from the different tissue samples obtained above using Trizol reagent (TIANGEN); cDNA was obtained by reverse transcription using the obtained total RNA as a template and the FastKing RT Kit (TIANGEN).

[0041] (3) Real-time PCR: Using the above cDNA as a template, the expression level of the OsZIP13 gene in the roots and leaves of different varieties under normal or alkaline stress conditions was detected by real-time PCR using OsZIP13-qRTS and OsZIP13-qRTAS primers.

[0042] The results are as follows Figure 3 As shown, under normal treatment conditions, the expression level of OsZIP13 in leaf tissues is relatively high. Under 50 mM NaHCO3 treatment, the expression level of OsZIP13 in both roots and leaves is significantly upregulated. Under salt-alkali stress conditions, the expression level in roots is about 15 times that of the control, and the expression level in leaves is about 1.5 times that of the control. This indicates that the expression of OsZIP13 gene is induced by salt-alkali stress, and its participation in the salt-alkali stress response is more significant in roots.

[0043] 4. Analysis of the expression pattern of the OsZIP13 gene under iron deficiency stress: (1) Treatment of plant materials: Select plump seeds of the Japanese rice variety Nipponbare. First, soak the seeds in water for 24 hours, then rinse them four times and place them on moist filter paper. Incubate them in the dark at 28°C for two days to promote rooting and germination. When the roots grow to about 2 cm, transfer them to a 96-well plate and place them in a container filled with Kimura B nutrient solution, so that the roots are immersed in the nutrient solution. Place them in an artificial climate chamber for cultivation. When the rice seedlings grow to 2 weeks old, transfer them to normal Kimura B nutrient solution and Kimura B nutrient solution without iron to treat them for iron deficiency stress. After 12 hours of treatment, select three rice seedlings with the same growth, and cut their root and leaf tissues as tissue samples to be tested. Quickly freeze them in liquid nitrogen and then store them at -80°C for later use.

[0044] (2) Extraction of total RNA and acquisition of cDNA: Total RNA was extracted from the different tissue samples obtained above using Trizol reagent (TIANGEN); cDNA was obtained by reverse transcription using the obtained total RNA as a template and the FastKing RT Kit (TIANGEN).

[0045] (3) Real-time PCR: Using the above cDNA as a template, the expression level of the OsZIP13 gene in the roots and leaves of different varieties under normal conditions or iron deficiency stress was detected by real-time PCR using OsZIP13-qRTS and OsZIP13-qRTAS primers.

[0046] The results are as follows Figure 4 As shown, under normal treatment conditions, the expression level of OsZIP13 in leaf tissues was higher than that in roots. Under iron deficiency treatment, the expression of OsZIP13 in both roots and leaves showed a downward trend, and the downregulation was significant. Under iron deficiency stress, the expression level of OsZIP13 in roots was about 0.6 times that of the control group, and the expression level of OsZIP13 in leaves was about 0.56 times that of the control group. This indicates that the expression of the OsZIP13 gene is induced by iron deficiency stress and participates in the iron deficiency stress response of rice.

[0047] 5. Analysis of the expression pattern of the OsZIP13 gene under zinc deficiency stress: (1) Treatment of plant materials: Select plump seeds of the Japanese rice variety Nipponbare. First, soak the seeds in water for 24 hours, then rinse them four times and place them on moist filter paper. Incubate them in the dark at 28°C for two days to promote rooting and germination. When the roots grow to about 2 cm, transfer them to a 96-well plate and place them in a container filled with Kimura B nutrient solution, immersing the roots in the nutrient solution. Place them in an artificial climate chamber for cultivation. When the rice seedlings reach 2 weeks of age, transfer them to normal Kimura B nutrient solution and Kimura B nutrient solution without zinc to treat them with zinc deficiency stress. After 12 hours of treatment, select three rice seedlings with the same growth rate, and cut their root and leaf tissues as test tissue samples. Quickly freeze them in liquid nitrogen and then store them at -80°C for later use.

[0048] (2) Extraction of total RNA and acquisition of cDNA: Total RNA was extracted from the different tissue samples obtained above using Trizol reagent (TIANGEN); cDNA was obtained by reverse transcription using the obtained total RNA as a template and the FastKing RT Kit (TIANGEN).

[0049] (3) Real-time quantitative PCR: Using the above cDNA as a template, the expression level of the OsZIP13 gene in the roots and leaves of different varieties under normal conditions or zinc deficiency stress was detected by real-time quantitative PCR using OsZIP13-qRTS and OsZIP13-qRTAS primers.

[0050] The results are as follows Figure 5 As shown, under normal treatment conditions, the expression level of OsZIP13 in leaf tissues was higher than that in roots. Under zinc deficiency treatment, the expression of OsZIP13 in both roots and leaves was significantly upregulated, with the most significant upregulation in roots, approximately 10.8 times that of the control group. In contrast, the expression level in leaves under zinc deficiency stress was approximately 3.2 times that of the control group. This indicates that the expression of the OsZIP13 gene is induced by zinc deficiency stress and is a zinc deficiency stress response gene in rice.

[0051] 6. Analysis of the expression pattern of the OsZIP13 gene under cadmium stress: (1) Treatment of plant materials: Select plump seeds of the Japanese rice variety Nipponbare. First, soak the seeds in water for 24 hours, then rinse them four times and place them on moist filter paper. Incubate them in the dark at 28°C for two days to promote rooting and germination. When the roots grow to about 2 cm, transfer them to a 96-well plate and place them in a container filled with Kimura B nutrient solution, immersing the roots in the nutrient solution. Place them in an artificial climate chamber for cultivation. When the rice seedlings reach 2 weeks of age, transfer them to normal Kimura B nutrient solution and Kimura B nutrient solution containing 50 μM CdCl2 for alkaline stress treatment. After 12 hours of treatment, select three rice seedlings with the same growth rate, and cut their root and leaf tissues as test tissue samples. Quickly freeze them in liquid nitrogen and then store them at -80°C for later use.

[0052] (2) Extraction of total RNA and acquisition of cDNA: Total RNA was extracted from the different tissue samples obtained above using Trizol reagent (TIANGEN); cDNA was obtained by reverse transcription using the obtained total RNA as a template and the FastKing RT Kit (TIANGEN).

[0053] (3) Real-time quantitative PCR: Using the above cDNA as a template, the expression level of the OsZIP13 gene in the roots and leaves of different varieties under normal conditions or cadmium ion stress conditions was detected by real-time PCR using OsZIP13-qRTS and OsZIP13-qRTAS primers.

[0054] The results are as follows Figure 6 As shown, under normal treatment conditions, the expression level of OsZIP13 in leaf tissue was higher than that in root tissue. After 12 h of treatment with 50 μM CdCl2, the expression level of OsZIP13 in root tissue decreased, approximately 0.75 times that of the control group. However, the expression level of OsZIP13 in leaf tissue increased after cadmium stress treatment, approximately twice that of the control group. This indicates that the expression of the OsZIP13 gene is inhibited in root tissue and induced in leaf tissue, participating in the rice response to cadmium ion stress. However, this study only selected one time point, and the expression level may have different responses at other time points. Example 2

[0055] This example illustrates the regulation of salt and alkali stress tolerance in transgenic rice plants.

[0056] 1. Identification of the zip13 mutant and its phenotypic analysis under alkaline stress: (1) Construction of CRISPR / Cas9 editing vector: Based on the cDNA sequence of the OsZIP13 gene, a targeting sgRNA of OsZIP13 was designed using CRISPR Primer Designer software and ligated into the CRISPR / Cas9 binary vector pYLCRISPR / Cas9Pubi-H. The nucleotide sequence of the targeting primer OsZIP13-LP, which is linked to the U3 promoter, is shown in SEQ ID No:9, and the nucleotide sequence of OsZIP13-RP is shown in SEQ ID No:10.

[0057] The target vector was transformed into Agrobacterium EHA105 and then introduced into the rice variety Nipponbare using Agrobacterium-mediated transformation. The oszip13 mutant target site is as follows: Figure 7 As shown in Figure A.

[0058] (2) Molecular identification of zip13 mutant plants: DNA was extracted from the zip13 mutant transgenic plant. Primers were designed at both ends of the targeting sgRNAs. Fragments containing the targeting region were expanded by PCR, and the targeting effect was analyzed by sequencing. The nucleotide sequence of the primer OsZIP13-Cas-InF for detecting the targeting effect is shown in SEQ ID No:11, and the nucleotide sequence of OsZIP13-Cas-InR is shown in SEQ ID No:12.

[0059] The results of the zip13 mutant targeting effect test are as follows: Figure 7 As shown in Figure B, two mutant lines were obtained. The zip13-1 mutant is a single base deletion, which leads to a shift in the OsZIP13 protein coding structure and premature translation termination. The other mutant, zip13-2, is a double base insertion, which also leads to a shift in the OsZIP13 protein coding structure and premature translation termination. Both mutations have a significant impact on the structure and function of the OsZIP13 protein. Therefore, these two mutant lines were selected for further research.

[0060] (3) Phenotypic analysis of zip13 mutant plants under alkaline stress: Select plump seeds of Nipponbare and zip13 mutant. First, soak the seeds in water for 24 hours. After the seeds show signs of sprouting, rinse them four times with clean water and place them on moist filter paper. One part is cultured normally as the control group, and the other part is treated with 30mM NaHCO3 for salt and alkali stress as the experimental group. The seeds are cultured in the dark at 28℃ for two days to promote rooting and sprouting. Then, phenotypic observation is carried out every day. When phenotypic differences appear, photos are taken and root length and plant height data are collected.

[0061] The results are as follows Figure 8As shown in Figure A, both wild-type (WT) and mutant plants can germinate normally, and salt-alkali stress does not affect their germination rate. However, compared with the wild type, the growth of the zip13 mutant is significantly inhibited under alkaline stress. Figure 8 As shown in Figures B and C, the statistical results of the shoot length and root length measurements also indicate that the growth of the zip13 mutant is significantly inhibited, suggesting that the zip13 mutant is more sensitive to salt and alkali stress.

[0062] 2. Identification of OsZIP13 overexpressing plants and their phenotypic analysis under alkaline stress: (1) Construction of plant expression vector for OsZIP13 gene: According to the instructions of the TransGen T1 gene cloning kit (pEASY-T1 Cloning Kit) from TransGen, the OsZIP13 gene cloned in Example 1 was ligated into the T vector. After correct sequencing, primers with restriction enzyme sites were designed, and the OsZIP13 gene was integrated into the plant expression vector pCAMBIA1302 by restriction enzyme digestion and ligation. Based on the multiple cloning site on pCAMBIA1302 and the restriction enzyme sites inside the gene, two restriction enzyme sites, Nco I (NEB) and Spe I (NEB), were selected. The stop codon was removed from the downstream primer to fuse the gene with the mGFP protein for expression. The nucleotide sequence of the upstream primer OsZIP13-En-F is shown in SEQ ID No:13, and the nucleotide sequence of the downstream primer OsZIP13-En-R is shown in SEQ ID No:14.

[0063] (2) Genetic transformation of rice: The recombinant vector pCAMBIA-OsZIP13 of the OsZIP13 gene was transformed into Agrobacterium tumefaciens LBA4404 using the freeze-thaw method. Positive transformants were obtained by PCR identification. The recombinant vector of the OsZIP13 gene was then transformed into the rice recipient material Nipponbare using Agrobacterium-mediated genetic transformation.

[0064] (3) Identification of transgenic rice plants: Regenerated tissue culture seedlings obtained after rice genetic transformation were screened for resistance to obtain resistant plants that could grow normally on 50 μM hygromycin (Sigma, 45520) medium. Total DNA was extracted from the resistant plants, and the transgenic plants were identified by PCR amplification of the resistance gene HPT. Some detection results are shown below. Figure 9 As shown, neither the wild-type (-) nor the negative control (H2O) showed the HPT resistance gene band, while HPT was detected in all the resistant plants tested, indicating that the target gene was successfully integrated into the genome of the recipient plant.

[0065] (4) Molecular identification of rice plants overexpressing OsZIP13: Total RNA was extracted from T3 generation transgenic OsZIP13 genotype rice seedlings and reverse transcribed to obtain cDNA. Using cDNA as a template and rice Ubi5 gene as an internal control, the expression level of OsZIP13 gene in transgenic plants was detected by real-time quantitative PCR, and OsZIP13 overexpression rice plants with Nipponbare cultivar background were obtained.

[0066] The results are as follows Figure 10 As shown, the expression level of OsZIP13 in overexpression lines 1–6 was significantly higher than that in the wild type (WT), all being more than 5 times the expression level of OsZIP13 in the wild type. Among them, the expression level of OsZIP13 was the highest in lines OE4, OE5, and OE1, which were 16.91, 15.31, and 11.75 times the expression level of OsZIP13 in the wild type, respectively. This indicates that the exogenous gene OsZIP13 has not only been successfully integrated into the rice genome, but can also be overexpressed normally in transgenic rice. Therefore, the three lines with high expression levels, OE4, OE5, and OE1, were selected for the next step of phenotypic analysis.

[0067] (5) Germination phenotype of OsZIP13 overexpression lines under alkali treatment: Seeds from plump wild-type Nipponbare and OsZIP13 overexpression lines were selected. First, the seeds were soaked in water at room temperature in the dark for 24 hours. Then, they were rinsed four times and placed on normally moistened filter paper (Control) and on moistened filter paper soaked in 30mM NaHCO3 for alkali stress treatment. After two days of dark incubation at 28℃ to promote rooting and germination, the seeds were transferred to a light incubator. The germination status, root length and shoot length were observed daily. When phenotypic differences were obvious, photos were taken and root and shoot lengths were counted. All experiments were repeated three times technically and three times biologically, with 90 plants of each line per experiment.

[0068] The results are as follows Figure 11 As shown in Figure A, under normal conditions, there was no significant difference in seed germination status between the wild-type (WT) and overexpression lines OE-1, OE-4, and OE-5, indicating that the introduced OsZIP13 gene did not affect the growth and development of rice during germination. Under 30 mM NaHCO3 stress, seed germination was slow in both the wild-type and overexpression lines, and root and shoot development were inhibited. Figure 11 As shown in Figure B, although there were no significant differences in germination rate and shoot length among different strains, root growth showed obvious differences. The root length of the overexpression strains was significantly higher than that of the wild type, indicating that they were less inhibited by alkali stress and had stronger alkali tolerance. At the same time, the shoot length of the overexpression strain OE-5 was also significantly higher than that of the wild type.

[0069] (6) Phenotype of seedlings overexpressing OsZIP13 under alkali treatment: Seeds of plump wild-type Nipponbare and OsZIP13 overexpression lines were selected. First, the seeds were soaked in water at room temperature for 24 hours in the dark. After rinsing four times, they were placed on moist filter paper and incubated in the dark at 28°C for two days to promote rooting and germination. When the roots reached approximately 2 cm in length, they were transferred to 96-well plates and placed in containers filled with Kimura B nutrient solution, ensuring the roots were submerged. The plates were then placed in an artificial climate chamber for cultivation. After two weeks of normal cultivation, alkali stress treatment was initiated. The plates were transferred to either a normal Kimura B nutrient solution (Control) or a Kimura B nutrient solution containing 50 mM NaHCO3 (pH 8.3) for further cultivation. Phenotypic changes were observed daily. All experiments were repeated three times technically and three times biologically, with 72 plants of each line per experiment.

[0070] The results are as follows Figure 12 As shown in Figure A, under normal conditions, there was no significant difference in seedling growth between wild-type (WT) and overexpression lines, indicating that the introduced OsZIP13 gene did not affect the growth and development of rice seedlings. Under 50 mM NaHCO3 stress, both wild-type and transgenic seedlings exhibited yellowing, wilting, and withering of older leaves, and their growth and development were inhibited. However, the transgenic plants showed better growth than the wild-type, exhibiting less chlorosis and wilting, and higher shoot length (shoot length during germination, and plant height during seedling stage). Figure 12 As shown in Figures B, C, and D, the fresh weight statistics indicate that the overexpression lines are significantly higher than the wild-type plants. Root length and plant height statistics also show that the overexpression lines are significantly higher than the wild-type plants, indicating that their inhibition under alkali stress is weaker. Meanwhile, as shown in Figures B, C, and D... Figure 13 As shown, chlorophyll content measurements indicated that under alkali stress treatment, chlorophyll levels decreased in all plants; however, the chlorophyll levels in transgenic plants were significantly higher than those in wild-type plants, indicating that transgenic plants have stronger alkali tolerance. Figure 14 As shown, the malondialdehyde (MDA) content of all strains increased under salt-alkali stress, while the MDA content of transgenic plants was significantly lower than that of wild-type plants, indicating that transgenic plants suffered less damage and had stronger alkali resistance.

[0071] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any way. Any simple modifications, alterations, and equivalent changes made to the above embodiments based on the inventive essence shall still fall within the protection scope of the present invention.

Claims

1. Use of an OsZIP13 gene, characterized in that, The OsZIP13 gene is used to regulate the salt and alkali stress tolerance of rice plants. Overexpression of the OsZIP13 gene in rice plants was used to enhance the salt and alkali stress tolerance of the rice plants. Or / and gene editing of the OsZIP13 gene in rice plants to reduce the salt and alkali stress tolerance of said rice plants; The nucleotide sequence of the coding region of the OsZIP13 gene is shown in SEQ ID No:1; the amino acid sequence of the coding region of the OsZIP13 gene is shown in SEQ ID No:

2. 2.The application of OsZIP13 gene according to claim 1, characterized in that, When the OsZIP13 gene in rice plants is overexpressed, the root length of rice plants under salt-alkali stress during the germination period is longer than that of wild-type rice plants. Under salt-alkali stress, the fresh weight, plant height, root length, and chlorophyll content of rice seedlings were all higher than those of wild-type rice plants, while the MDA content was lower than that of wild-type rice plants.

3. The application of the OsZIP13 gene according to claim 1, characterized in that, When the OsZIP13 gene in rice plants was edited, oszip13 mutant rice plants were obtained. Under salt-alkali stress, the plant height and root length of the oszip13 mutant rice plants were both lower than those of wild-type rice plants.

4. The application of the OsZIP13 gene according to claim 1, characterized in that, An overexpression vector for the OsZIP13 gene was constructed to cultivate transgenic rice plants with high tolerance to salt and alkali stress.

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