Application of low-potassium rice transcription factor KTR1 in reducing the absorption and transport of cesium in rice.

Abstract

This invention discloses the application of the rice low-potassium repressive transcription factor KTR1 in reducing the absorption and transport of the heavy metal cesium in rice. A low-potassium repressive transcription factor encoding a gene, KTR1, is shown in SEQ ID NO.1. This transcription factor can reduce the absorption and transport of the heavy metal cesium (Cs) in rice by inhibiting the expression of OsHAK1. + This invention relates to applications in the absorption and transport of cesium (Cs). Through extensive experiments, this invention has discovered the biological function of the low-potassium transcription factor KTR1 in rice. Overexpression of this gene reduces the absorption of the heavy metal cesium (Cs) in rice. + The intake and accumulation of ).

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology and relates to the method of reducing the rice's resistance to the heavy metal cesium (Cs) by inhibiting the expression of the low-potassium transcription factor KTR1. + Applications in the absorption and transport of . Background Technology

[0002] Potassium (K) + K+ is the most abundant essential cation in plants, playing several key roles, including maintaining ionic charge balance, regulating turgor pressure, and regulating enzyme activity (Anschütz et al., 2014; Li et al., 2018). As a highly mobile cation, K+... + Promote the cycling of photosynthetic carbon from source to sink, as well as various other nutrients, especially nitrates and amino acids, to improve food yield and nutrient use efficiency (Marschner et al., 1996; Peng et al., 2023).

[0003] Cesium (Cs) + It is toxic to plants, belongs to group I of alkali metals, and is similar to potassium (K). + They have similar chemical properties. By increasing the potassium content in the soil... + Levels can alleviate Cs + The toxicity of Cs (Shaw & Bell, 1991; Zhu & Smolders, 2000). Studies have shown that plant Cs... + Toxicity is caused by K + and Cs + Between K + Competition for binding sites with proteins essential for their binding, not just K. + Caused by starvation (Hampton et al., 2005). Human exposure to radioactive Cs + The primary cause of serious health hazards is through the consumption of contaminated agricultural products (White & Broadley, 2000; Zhu & Smolders, 2000; Kinoshita et al., 2011). Therefore, reducing the edible portion of crops is crucial for protecting Cs. + The absorption and accumulation of these nutrients are crucial for public health security.

[0004] High Affinity K + Transporter protein (HAK) in external low-kJ + Mediating K under conditions below 0.2 mM +They play a central role in K uptake (Wang & Wu, 2013; Nieves-Cordones et al., 2016). Studies of various HAK members in plant species have revealed their role in K uptake. + Multifaceted roles in absorption and translocation, salt tolerance, osmotic potential regulation, and root and stem development regulation (Li et al., 2018). Most of the identified Cs... + Transport proteins are all related to K + Transporters are directly or indirectly involved, particularly members of the HAKs group (Kobayashi et al., 2010; Nieves-Cordones et al., 2017; Rai et al., 2017; Yamaki et al., 2017; Ródenas et al., 2018). Recently, two ATP-binding proteins, ABCG37 and ABCG33, have been shown to act as K transporters in Arabidopsis roots. + Non-dependent Cs + The absorber has redundancy (Ashrafet al., 2021).

[0005] OsHAK5 participates in K in rice + The acquisition and transport of nutrients from root to stem, in low K + Under certain conditions, ATP-dependent auxin influx alters the morphology and structure of rice (Yang et al., 2014; Yang et al., 2020), but for Cs... + It plays almost no role in the absorption of K (Uchiyama et al., 2022). In contrast, OsHAK1 plays a significant role in the absorption of K+. + Maintain K within the concentration range + These are important genes that mediate growth (Chen et al., 2015; Chen et al., 2017; Li et al., 2022), and are also crucial for the absorption and transport of Cs in rice. + The main pathway. Knocking out OsHAK1 significantly reduced the rice root system's resistance to Cs. + Absorption rate of Cs in seeds and stems + Concentration (Nieves-Cordones et al., 2017; Rai et al., 2017). Interestingly, OsHAK1 showed poor differentiation of Cs. + and K + Absorption capacity, but from very low external concentrations to high Cs concentrations. + Obtaining Cs from contaminated soil +Their abilities are very high (Nieves-Cordones et al., 2017).

[0006] Most identified HAK genes are regulated at the transcriptional level, and recent advancements have yielded significant progress in elucidating the regulatory mechanisms of HAK transporters at both the transcriptional and post-translational levels (Wang & Wu, 2017; Li et al., 2018; Ragel et al., 2019; Wang et al., 2021; Johnson et al., 2022). Using a gene activation marker method with the AtHAK5 promoter fused to a luciferase reporter (LUC), an Arabidopsis AP2 / ERF transcription factor, RAP2.11 (associated with AP2 11), was identified as a direct activator of AtHAK5. This transcription factor also regulates the regulation of low-K pathways in Arabidopsis roots. + The signaling pathway involves the expression of numerous genes (Kim et al., 2012). Furthermore, the transcription factor MYB77 actively regulates AtHAK5 expression by binding to the HAK5 promoter and enhances high-affinity K... + Intake (Feng et al., 2021). Furthermore, the auxin-responsive factor ARF2 can be absorbed at high K levels. + Supply negatively regulates AtHAK5 expression to reduce AtHAK5-mediated K + The energy cost of intake (Zhao et al., 2016). These studies indicate that plants require multiple regulatory factors to activate or inhibit K+. + Transporter proteins to adapt to different external K + Supply. Therefore, in this study, we identified a low-potassium-inhibited AP2 / ERF transcription factor, KTR1, in rice that reduces rice's resistance to Cs by directly binding to the promoter of OsHAK1 and inhibiting OsHAK1 expression. + The absorption and translocation of cesium (Cs) provides a basis for the cultivation of heavy metal resistant cesium (Cs). + The newly planted rice varieties contaminated with the contaminated rice provide new target genes. Summary of the Invention

[0007] The objective of this invention is to provide a rice low-potassium transcription factor KTR1 that reduces the susceptibility of rice to the heavy metal cesium (Cs) by inhibiting the expression of OsHAK1. + Its application in the absorption and transport of cesium (Cs) is mainly that self-promoter overexpression materials can increase the absorption of heavy metal cesium (Cs) in rice seedlings. + Tolerance to stress.

[0008] The objective of this invention is achieved through the following techniques:

[0009] Low potassium levels in rice inhibit the transcription factor KTR1, reducing the rice's susceptibility to the heavy metal cesium (Cs). + Application in the absorption and transport of ), the CDS sequence of the rice low potassium response transcription factor gene KTR1 is shown in SEQ ID NO.1 (SEQ ID No.1: CDS sequence of KTR1).

[0010] As a preferred embodiment of the present invention, overexpression of the rice low-potassium repressor transcription factor gene KTR1 reduces the resistance of rice seedlings to the heavy metal cesium (Cs). +The intake and accumulation of ).

[0011] The recombinant expression vector overexpressing the rice inhibitory response transcription factor KTR1 reduced the rice's susceptibility to the heavy metal cesium (Cs). + Applications in intake and accumulation.

[0012] As a preferred embodiment of the present invention, the recombinant expression vector is obtained by inserting the rice low-potassium repressive transcription factor KTR1 into the KpnI and SalI restriction sites of the starting vector pCAMBIA1305, then using EcoRI and KpnI double enzymes to remove the Ubiquitin promoter and inserting it into the KTR1 gene's own promoter.

[0013] The beneficial effects of this invention are:

[0014] 1. In this invention, it was discovered for the first time that a recombinant expression vector constructed from the rice low-potassium repressor transcription factor KTR1 was transfected into wild-type rice (Oryza sativa. ssp. cv. Japonica) using Agrobacterium-mediated rice transgenic technology. The absorption and translocation of the heavy metal cesium in the seedling stage of the KTR1 overexpression material was significantly reduced compared with that of wild-type rice.

[0015] 2. The KTR1 gene in this invention is derived from rice, and its promoter is also an upstream sequence of the KTR1 gene. It is not a foreign gene, therefore it has biosafety. The constructed rice low-potassium repressor transcription factor KTR1 plant expression vector can be directly used for Agrobacterium-mediated plant genetic transformation to obtain a vector that reduces the susceptibility of rice seedlings to the heavy metal cesium (Cs). + ( ) new germplasm absorbed and transported. Attached Figure Description

[0016] Figure 1 KTR1 expression was downregulated by low potassium, ethylene, and H2O2 stress.

[0017] Figure A: KTR1 gene affected by low potassium (0.05 mM K) + Figure B: Expression levels of the KTR1 gene in the roots and aboveground parts after stress; Figure B: Expression levels of the KTR1 gene in the roots of rice at different time points (0.25h, 2h and 6h) after stress with 10 mM H2O2 and 0.1 mM ethylene.

[0018] Figure 2 Expression of OsHAK1 in different tissues of KTR1-Ox material under normal and low potassium conditions

[0019] Figure A: OsHAK1 expression levels in different tissues of WT and KTR1 overexpression materials under normal potassium conditions; Figure B: OsHAK1 expression levels in different tissues of WT and KTR1 overexpression materials under low potassium conditions.

[0020] Figure 3 KTR1 self-promoter overexpression and corresponding wild-type material phenotypes after cesium treatment

[0021] Figure A: Phenotypes of plants overexpressing WT and KTR1 after cesium treatment; Figure B: Dry weight of roots and shoots of WT and KTR1 overexpressing materials after cesium treatment; Figure C: Cesium content in roots and shoots of WT and KTR1 overexpressing materials after cesium treatment; Figure D: Total cesium content in roots and shoots of WT and KTR1 overexpressing materials after cesium treatment.

[0022] Figure 4 KTR1 inhibits OsHAK1 expression in vivo and in vitro by directly binding to the OsHAK1 promoter.

[0023] Figure A: Schematic diagram of the OsHAK1 promoter region structure, showing the location of the GCCGCC element; Figure B: Dual-luciferase transient expression experiment demonstrating the transcriptional repression of OsHAK1 by KTR1; Figure C: ChIP-qPCR experiment demonstrating the binding of KTR1 to the OsHAK1 promoter in rice; Figure D: EMSA experiment demonstrating the binding ability of KTR1 to the OsHAK1 promoter in rice in vitro.

[0024] Figure 5 The map of the pCAMBIA1305 expression vector.

[0025] Figure 6 ,Dual luciferase vector map.

[0026] Figure 7 pMAL-c5x vector spectrum. Detailed Implementation

[0027] Example 1. Identification of the response of rice KTR1 to environmental stresses such as low potassium, ethylene, and H2O2 using RT-qPCR technology.

[0028] To investigate the response of the KTR1 gene to environmental stresses of low potassium, ethylene, and H2O2, seedlings (Nipponbare wild type) were incubated in 1 / 2 MS medium. Ten days after incubation, robust and uniformly growing seedlings were transferred to an 8-liter transport box and cultured in normal nutrient solution for two weeks, with the nutrient solution changed every two days. Afterward, a low potassium (0.05 mM K) incubation period was implemented. +Root and aboveground samples were collected after 4 weeks of treatment. Root samples were also collected after treatment with 10 mM H2O2 and 0.1 mM ethylene for 0.25 h, 2 h, and 6 h, respectively.

[0029] Total RNA was extracted from different tissue samples using Trizol reagent (Invitrogen), followed by removal of genomic DNA and reverse transcription into cDNA. The synthesized cDNA was used as a template for qRT-PCR. Four biological replicates were set up for this experiment, and the primers used for quantification are shown in Table 1.

[0030] Table 1. Primer sequences for OsActin and KTR1 used in qRT-PCR

[0031]

[0032] The experimental results showed that KTR1 expression was downregulated by low potassium, ethylene, and H2O2 stress. When 0.05 mM K... + After 4 weeks of treatment, the expression level of KTR1 in rice roots was approximately 1 / 5 of that in WT (whole wheat rice). Figure 1 A), while the aboveground parts showed no significant difference from the wild type ( Figure 1 A). When treated with 10 mM H2O2 and 0.1 mM ethylene for 6 h, KTR1 expression was significantly downregulated, approximately half that of WT. Figure 1 B).

[0033] Example 2. Construction of KTR1 self-promoted overexpression materials

[0034] To obtain KTR1 self-promoter overexpression material, the pCAMBIA1305.1-35ST vector was double-digested with KpnI and SalI. Figure 5Using wild-type cDNA from Nipponbare as a template, the CDS sequence of KTR1 without a stop codon was obtained by PCR amplification (primers: KTR1-F / R, see Table 2). Next, the KTR1 CDS sequence was ligated to a 3×Flag tag sequence by PCR (primers: Overlapping-F / R, see Table 2). The product was ligated into the enzyme-digested pCAMBIA1305.1-35ST linear vector and transformed into DH5α competent cells. After verification by colony PCR and Sanger sequencing, the intermediate vector KTR1-Flag-1305 was obtained. To obtain self-promoter overexpression material, the KTR1-Flag-1305 intermediate vector was first digested with EcoRI and KpnI to remove the Ubiquitin promoter. Next, using wild-type gDNA of Nipponbare rice as a template, the first 2178 bp of the KTR1 ATG promoter sequence was amplified by PCR (primers: ProKTR1-F / R, see Table 2). The KTR1 promoter sequence was then ligated into the intermediate vector KTR1-Flag-1305 via homologous recombination to obtain the final vector KTR1-Ox. The final vector plasmid was transformed into DH5α competent cells. Positive transformants containing the KTR1 promoter were obtained after colony PCR and Sanger sequencing verification. The plasmid of these transformants was electroporated into Agrobacterium EHA105, and positive clones were selected after antibiotic screening. Plasmid extraction and coliform transfection were performed for verification. Verified positive clones were used to infect Nipponbare rice callus to obtain corresponding transgenic materials.

[0035] Table 2 Primer sequences for KTR1-Ox

[0036]

[0037] Note: Underlined bases are enzyme cleavage sites (BamHI: GGATCC; KpnI: GGTACC; SalI: GTCGAC; EcoRI: GAATTC).

[0038] Example 3. Identification of KTR1 overexpression materials

[0039] Nipponbare wild-type and overexpression (T1 generation) seedlings were water-grown. After 10 days, the overexpression material was identified as GUS positive. Seedlings with consistent growth were cultured in normal nutrient solution for two weeks. Root and leaf samples were collected to extract RNA. cDNA was obtained by reverse transcription and then the overexpression effect was identified using semi-quantitative primers (Table 3).

[0040] Table 3. Primer sequences for OsActin and KTR1 used in RT-PCR

[0041]

[0042] Example 4: Identification of OsHAK1 expression level in KTR1-Ox material using RT-qPCR technology

[0043] To detect the expression of the OsHAK1 gene in KTR1 overexpression materials, wild-type Nipponbare seedlings and KTR1 overexpression materials were cultured in 1 / 2 MS medium. Ten days later, seedlings with uniform growth were selected and cultured in normal nutrient solution and low-potassium nutrient solution for 4 weeks, respectively. Root and shoot samples were then collected for quantitative PCR to detect the expression level of OsHAK1. The OsHAK1 quantitative primers are shown in Table 4. The results showed that, under both normal and low-potassium conditions, the expression level of OsHAK1 in the overexpression materials was significantly lower than that in the wild type, decreasing by approximately 4-6 times. Figure 2 AB).

[0044] Table 4 Primer sequences for OsActin and OsHAK1 used in RT-qPCR

[0045]

[0046] Example 5: KTR1 overexpression materials and corresponding wild-type Cs + Effects of absorption

[0047] To determine the effects of KTR1 overexpression materials and wild-type rice seedlings on Cs + The absorption and translocation of K+ will be carried out by culturing 10-day-old rice seedlings with uniform growth in a solution containing 1 mM K+. + It was grown in IRRI solution for 8 weeks, and then transferred to a solution containing 1 mM K. + and 0.2mM Cs + Rice was treated with IRRI liquid for 6 days. Rice phenotype was observed and photographed. Root and aboveground samples were collected separately, dried, weighed, and Cs were detected using ICP-MS. + concentration.

[0048] The experimental results showed that, compared with the wild type, the aboveground dry weight of the KTR1-Ox strain was significantly reduced ( Figure 3 B) and Cs + The content was significantly reduced ( Figure 3 C), therefore, the total cesium content in the aboveground parts of the KTR1-Ox strain was also significantly reduced (C). Figure 3 D). In summary, KTR1 inhibits the absorption and accumulation of cesium in rice by directly binding to the OsHAK1 promoter and suppressing OsHAK1 expression, thereby enhancing the crop's resistance to the heavy metal cesium and providing a new target gene for breeding new cesium-resistant germplasm varieties.

[0049] Example 6: ChIP-qPCR verification of GCC-box binding of KTR1, KTR2 and OsHAK1 promoters

[0050] The materials used in the ChIP-qPCR experiment were KTR1 self-promoter overexpression lines.

[0051] The procedure was performed using the EpiQuik™ Plant ChIP Kit (Epigentek). Approximately 1 g of transgenic seedlings was cross-linked with 1% formaldehyde for 10 min, and the cross-linking reaction was terminated with glycine. The cross-linked DNA-protein complex was then disrupted by sonication, and anti-Flag antibody (CST Rabbit anti) was added. qPCR quantitative primers were designed based on the OsHAK1 promoter sequence (Table 5), and qPCR analysis was performed using the immunoprecipitated genomic DNA fragments. Due to the close proximity of the six GCC-Boxes on the OsHAK1 promoter, we selected a 256 bp fragment of the OsHAK1 promoter for the ChIP-qPCR experiment, which included the aforementioned six GCC-Boxes.

[0052] Table 5. Primers for ChIP-qPCR Quantitative Analysis

[0053]

[0054] We found that KTR1 and the OsHAK1 promoter can bind in vitro using transient expression in tobacco, dual-luciferase transient expression, and EMSA gel retardation assays. However, these experiments were insufficient to demonstrate their binding in rice. Therefore, we further verified the specific binding of the transcription factor KTR1 to the OsHAK1 promoter using ChIP-qPCR. The results showed that after immunoprecipitation, the DNA enrichment at the OsHAK1 promoter GCC-Box in the KTR1-Ox transgenic line was approximately 4-6 times higher than that in the Pro:Ubi-Flag negative control group. This indicates that the KTR1 transcription factor can directly bind to the OsHAK1 promoter GCC-Box in vivo. Figure 3 (B) However, because the six GCC-Boxes on the OsHAK1 promoter are too close together, we cannot determine whether the transcription factor KTR1 binds to any specific GCC-Box on the OsHAK1 promoter or to all of them.

[0055] Example 7: Dual-luciferase transient expression assay

[0056] The pGreenII 0800-LUC vector was double-digested using SalI and HindIII restriction sites (see diagram). Figure 6The 3010 bp promoter sequence upstream of the OsHAK1 coding region was cloned into the pGreenII 0800-LUC vector to drive the expression of the luciferase (LUC) gene. Simultaneously, the pGreenII 62-SK vector was digested with BamHI and SalI to clone the 801 bp CDS sequence of KTR1 into the pGreenII 62-SK vector as the effector vector (see diagram). Figure 6 The primers used to construct the vector are shown in Table 6. Finally, the correctly sequenced strains and plasmids were stored at -80℃ and -20℃ respectively for later use.

[0057] Table 6 Primer sequences for the construction of dual-luciferase vectors

[0058]

[0059] Note: Underlined bases are enzyme cleavage sites (NdeI: CATATG; EcoRI: GAATTC).

[0060] The experiment was divided into two groups: empty-62SK + OsHAK1-08 and KTR1-62SK + OsHAK1-0800. Group 1 served as the negative control group, and Group 2 was the experimental group. PEG-mediated plasmid transformation was used, with each plasmid co-transformed into rice protoplasts. After transient transformation and incubation in the dark for approximately 16-20 h, dual-luciferase activity was detected using a dual-luciferase reporter assay kit (Promega). The ratio was used to reflect the degree of binding between transcription factors and downstream gene promoters. The results showed that in high K... + Under these conditions, after KTR1 co-transformed with OsHAK1-LUC, the LUC / REN ratio was significantly lower than that of the control group ( Figure 3 C).

[0061] Example 8 Construction of prokaryotic expression vector

[0062] NdeI and EcoRI (Table 7) were added to both ends of the primers for amplifying the full-length KTR1 gene. Amplification was performed using plasmid DNA containing the full-length CDS sequence of the KTR1 gene as a template. The PCR product was recovered, purified, and ligated into the pESAY-Blunt cloning vector. The vector was then transfected into the colon to verify the correct transformant plasmid DNA and pMAL-c5x vector. Figure 7 The cells were digested with NdeI and EcoRI, the products were recovered and purified, ligated with T4 enzyme, and the ligation products were transformed into Rosetta competent cells. Positive clones were screened with Amp antibiotic and selected. Plasmids were extracted, digested with enzymes, and sequenced for verification. The verified positive clones were used for prokaryotic protein extraction.

[0063] Table 7 Primer sequences for constructing the OsKTR1 prokaryotic expression vector

[0064]

[0065] Note: Underlined bases are enzyme cleavage sites (NdeI: GGTACC; EcoRI: GAATTC).

[0066] Example 9 Extraction and purification of prokaryotic expressed proteins

[0067] 1. Inducible expression of prokaryotic proteins

[0068] ①Transfect Rosetta bacteria with the target gene or empty vector, and incubate overnight in test tubes at 37 ℃;

[0069] ② On the second day, add 1.5 ml of bacterial culture to a 150 mL LB bottle containing the appropriate antibiotic (the system can be expanded as needed), and shake at 37 ℃ until OD is reached. 600 Approximately 0.6-0.8;

[0070] ③ Add 200 μl of 0.1 M IPTG (isopropyl-β-d-thiogalactopyranoside, final concentration 0.1 mM), place the bacterial culture in a shaker at 16~20 ℃ and 200 rpm for 10-16 h;

[0071] ④ Centrifuge 50 ml centrifuge tubes at 8000 rpm for 5-10 min at 4 ℃;

[0072] ⑤ Resuspend the bacterial cells in 20 ml PBS solution and transfer the solution to a 60 ml glass bottle (to facilitate subsequent sonication).

[0073] ⑥ Add 100 μl of 100 mg / ml lysozyme to 20 ml of bacterial culture and place on ice for 20-30 min. At this time, you can also add 400 μl of PMSF (0.1 M stock solution, 2 mM final concentration) and 1 / 4 tablet of protease inhibitor (Cocktail) to prevent protein degradation.

[0074] ⑦Ultrasonic fragmentation: 10 ϕ setting, 2~3 s, 2 s, 12~15 min;

[0075] 2. Purification of prokaryotic proteins

[0076] ① Column packing: Pipette 2.5 ml of resin into the purification column, and discard the alcohol solution that was used to preserve the resin before the column is flushed away;

[0077] ② Column pretreatment: First, treat with 3 column volumes of water, then with 5 column volumes of column buffer.

[0078] ③ At 4 ℃, the crude protein sample after ultrasonic disruption flows through the column, with the flow rate controlled at < 60 cm / h (gravity flow) (can be operated in a 4 ℃ refrigerator);

[0079] ④ Wash the column with 10 column volumes of column buffer (25 ml);

[0080] ⑤ Elute the target protein with 3-5 ml of Elution Buffer;

[0081] 3. Determination of protein concentration

[0082] ① Based on the number of samples, prepare an appropriate amount of BCA working solution by adding 50 volumes of BCA reagent A to 1 volume of BCA reagent B (50:1) and mix thoroughly (BCA working solution is stable at room temperature for 24 hours).

[0083] ② Dissolve the BSA protein standard to a concentration of 2 μg / μl and prepare a mark, as follows:

[0084]

[0085] ③ Add 25 μl of protein sample or BSA standard to the ELISA plate;

[0086] ④ Add 200 μl of the prepared BCA working solution, mix thoroughly, cover and incubate at 37 ℃ for 30 min;

[0087] ⑤ Remove the ELISA plate and allow it to cool to room temperature;

[0088] ⑥ Measure the absorbance at a wavelength of 562 nm using an ELISA reader;

[0089] ⑦ Create a standard curve and calculate the protein concentration.

[0090] Example 10 Gel Electrophoresis Mobility Assay (EMSA)

[0091] This experiment used the EMSA kit (Thermo Scientific, catalog number 20148).

[0092] ① Prepare a 4% polyacrylamide gel and perform pre-electrophoresis;

[0093] ② Add samples according to the table below to carry out the binding reaction, and then let it stand at room temperature for 20 min. The mutation probe is to mutate GCCGGC in the OsHAK1 promoter P3 to TTTTTT.

[0094]

[0095] ③ Add 5 μL of 5X loading buffer to the reaction solution, pipette and mix well, add the sample, and electrophoresis at 100 V until the blue dye migrates to 2 / 3 or 3 / 4 of the gel.

[0096] ④ Transfer membrane

[0097] a. A nylon film slightly larger than the adhesive block is immersed in 0.5X TBE for at least 10 minutes;

[0098] b. Sandwich the nylon membrane, non-degrading gel, absorbent cardboard, and sponge in sequence and place them into a clean electrophoresis transfer apparatus. Pre-cool 0.5X TBE to approximately 10 °C;

[0099] c. Transfer the membrane at 100 V for 30~60 min.

[0100] d. After the transfer is complete, place the nylon membrane with the bromophenol blue side on a dry paper towel to allow the buffer solution on the membrane surface to be absorbed into the membrane.

[0101] ⑤ UV-light cross-linker (120 mJ / cm) 2 , 45~60 s;

[0102] ⑥ Probes for chemiluminescence detection of biotin-labeled molecules

[0103] a. Use a 37-50℃ water bath to block buffer and washing solution until the small particles inside are completely dissolved;

[0104] b. 20 ml blocking solution, gently shake the blocking membrane for 15 min;

[0105] c. Discard the blocking solution, add 20 ml of blocking solution containing 66.7 μl of horseradish peroxidase avidin, and gently shake for 15 min;

[0106] d. Transfer the membrane to a new container and gently rinse 5 times with 20 ml of washing solution, 5 min each time;

[0107] e. Transfer the membrane to a new container, add 30 ml of equilibration buffer, shake gently for 5 min, and prepare the color development working solution (6 ml of enhancement solution + 6 ml of substrate solution).

[0108] f. Remove the membrane, carefully place the edges of the membrane on a paper towel to absorb excess solution, and place the membrane in a clean container or on a clean plastic sheet placed on a flat surface;

[0109] g. Pour 12 ml of colorimetric working solution onto the membrane, ensuring the solution completely covers the membrane surface, and let it stand at room temperature for 5 min;

[0110] h. Wrap the membrane in plastic film to avoid air bubbles and wrinkles, and take a picture using an instrument with a CCD camera. Adjust the exposure time as needed.

[0111] EMSA results showed that KTR1 can specifically bind to the biotin-labeled OsHAK1 probe fragment ( Figure 3 D), after DNA dot mutation of the GCC-box on this fragment, this binding cannot occur ( Figure 3 D). Meanwhile, the negative control MBP empty vector protein and the biotin-labeled OsHAK1 probe fragment cannot bind ( Figure 3 D). Based on the above results, we hypothesize that KTR1 may negatively regulate its expression by binding to the GCC-box on the OsHAK1 promoter.

Claims

1. Overexpression of rice low-potassium repressor transcription factor KTR1 Reducing the exposure of rice to the heavy metal cesium (Cs) + Application in the uptake and accumulation of the rice low-potassium repressor transcription factor KTR1 The CDS sequence is shown in SEQ ID NO.

1.

2. The application according to claim 1, characterized in that, Overexpression of rice low-potassium repressor transcription factor KTR1 It will reduce the absorption of the heavy metal cesium (Cs) during the rice seedling stage. + The intake and accumulation of.

3. The application according to claim 1, characterized in that, Overexpression of rice low-potassium repressor transcription factor KTR1 By inhibiting OsHAK1 The expression of cesium Cs reduces the resistance of rice seedlings to the heavy metal Cs. + The intake and accumulation of.

4. Contains the rice low-potassium transcriptional inhibitor as described in claim 1 KTR1 Recombinant expression vectors reduce the susceptibility of rice seedlings to the heavy metal cesium (Cs). + Application in the intake and accumulation of.

5. The application according to claim 4, characterized in that, The recombinant expression vector is the rice low-potassium repressor transcription factor described in claim 1. KTR1 Inserted into the launch carrier pCAMBIA1305 KpnI and SalI After the enzyme cleavage site is used EcoRI and KpnI Double enzyme excision Ubiquitin Startup and Insertion KTR1 Obtained from the gene's own promoter.

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