A method for regulating plant selective uptake of cadmium and manganese by dephosphorylation

Abstract

This application relates to a method for regulating the selective uptake of cadmium and manganese in plants through dephosphorylation. STNP-mediated phosphorylation is crucial for the selective uptake of cadmium and manganese in Solanaceae NRAMP5. A mutation mimicking nonphosphorylation in NRAMP5 reduces cadmium uptake without affecting manganese uptake.

Description

Technical Field

[0001] This application belongs to the field of genetic engineering technology, specifically relating to a method for regulating the selective absorption of cadmium and manganese in plants through dephosphorylation. Background Technology

[0002] Cadmium (Cd) pollution in the environment poses a significant threat to food safety and human health due to its high toxicity and ease of absorption into the food chain through plant roots. Researchers have identified genes responsible for cadmium accumulation in plants and knocked them out to reduce cadmium accumulation. However, while these genes promote cadmium absorption, they may also play a role in the absorption of essential nutrients such as manganese. For example, knocking out OsNRAMP5 in rice significantly reduces cadmium accumulation, but also reduces manganese accumulation, leading to losses in rice growth and yield. Therefore, a dilemma exists between reducing cadmium absorption and stabilizing the demand for essential metals. Summary of the Invention

[0003] To address the aforementioned technical problems, this application provides a method for reducing cadmium absorption in plants, comprising dephosphorylating the N-terminus of the NRAMP5 protein in the plant.

[0004] In some embodiments, the N-terminus of the plant's NRAMP5 protein is the first 41 amino acids of the N-terminus with reference to SEQ ID NO.1, preferably the first 30, 31, 32, 33, 34, or 35 amino acids of the N-terminus.

[0005] In some embodiments, the dephosphorylation treatment targets amino acids 27-28 or 27-30 of the sequence referenced in SEQ ID NO.1.

[0006] In some embodiments, dephosphorylation is performed on the N-terminus of the plant NRAMP5 protein, targeting two consecutive amino acids of the ST sequence or four consecutive amino acids of the STNP sequence.

[0007] In some embodiments, dephosphorylation is performed by nonphosphorylating two consecutive amino acids of ST or four consecutive amino acids of STNP at the N-terminus of the plant NRAMP5 protein.

[0008] In some embodiments, dephosphorylation is performed by deleting two consecutive amino acids of ST or four consecutive amino acids of STNP from the N-terminus of the plant NRAMP5 protein.

[0009] This application also provides a method for increasing cadmium absorption in plants, comprising site-specifically reintroducing two consecutive amino acids of ST or four consecutive amino acids of STNP into the N-terminus of the NRAMP5 protein of the plant, wherein the N-terminus of the NRAMP5 protein of the plant does not contain two consecutive amino acids of ST or four consecutive amino acids of STNP.

[0010] In some implementations, the location of the site-specific replacement is immediately after the 26th amino acid in the sequence referenced by SEQ ID NO.1.

[0011] In some embodiments, the point of refill is located immediately after the N-terminal LPS of the NRAMP5 protein of the plant, three consecutive amino acids.

[0012] This application also provides a method for predicting the cadmium uptake capacity of plants, including the step of detecting whether the N-terminus of the plant's NRAMP5 protein has two consecutive ST amino acids or four consecutive STNP amino acids; if it has, the plant is predicted to have a strong cadmium uptake capacity; if it does not, the plant is predicted to have a weak cadmium uptake capacity.

[0013] In some embodiments, the plant is a monocotyledonous or dicotyledonous plant, preferably a plant of the Solanaceae family, such as one or more of black nightshade, tomato, potato, eggplant or pepper.

[0014] The beneficial effects of this application are: by discovering and utilizing the substrate selectivity of NRAMP5, the accumulation of harmful metals (such as cadmium) is minimized without affecting the plant's acquisition of essential metal elements for growth (such as manganese). Attached Figure Description

[0015] Figure 1 Identification of cadmium absorption capacity of SaNRAMP5, StNRAMP5, SlNRAMP5, CaNRAMP5 and SmNRAMP5.

[0016] Figure 2 Growth curves of Δycf1 yeast cells transformed with different NRAMP5s under 0 and 8 μM CdCl2.

[0017] Figure 3 Phylogenetic tree of NRAMP5 protein.

[0018] Figure 4 The effect of replenishing STNP or AANP residues in StNRAMP5 and SlNRAMP5 on their cadmium uptake capacity.

[0019] Figure 5 Growth curves of yeast cells with different Δycf1 transformed with different NRAMP5s under 0 and 8 μM CdCl2.

[0020] Figure 6 The effect of dephosphorylation of SaNRAMP5 on cadmium absorption capacity.

[0021] Figure 7 Characterization of Mn absorption capacity of SaNRAMP5, StNRAMP5, SlNRAMP5, CaNRAMP5 and SmNRAMP5.

[0022] Figure 8 : Characterize the effect of the phosphate state of NRAMP5s on manganese absorption capacity. Detailed Implementation

[0023] To illustrate the universal design concept of this application, specific experimental parameters are used as examples below, but this should not be used as a reason to limit the scope of protection of this application.

[0024] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0025] Example 1: Experimental Design Ideas and Methods

[0026] Using total cDNA from the roots of tomatoes, potatoes, eggplants, and peppers as templates, PCR primers were designed to amplify the CDS sequences of SaNRAMP5, SmNRAMP5, StNRAMP5, SlNRAMP5, and CaNRAMP5, respectively. The PCR program was as follows: 95℃ pre-denaturation for 3 min, 95℃ denaturation for 30 s, 56℃ annealing, 72℃ annealing and extension for 1 min 20 s, 35 cycles, followed by 72℃ for 7 min. The amplified PCR products were detected by 1% agarose gel electrophoresis, and the fragment size was approximately 1600 bp. The target PCR products were separated by agarose gel electrophoresis and then recovered by gel excision. The yeast expression vector pDR196 was digested and purified using EcoR1 and Xho1. The linearized plasmid and the purified PCR products of each gene were subjected to homologous recombination: the system was 5 μL, containing 1 μL of linearized vector and 4 μL of purified PCR product, and then transformed at 37 degrees for 1 h. Single clones were picked and sequenced. The correct single clones were preserved and the plasmids were named StNRAMP5-pDR196, SlNRAMP5-pDR196, SmNRAMP5-pDR196 and CaNRAMP5-pDR196. Point mutations were constructed using pre-constructed pDR196-ligated vectors as templates, followed by overlap extension PCR. Specifically, one set of primers was used: SaNRAMP5-pDR196F and SaNRAMP5(AANP)-pDR196R. Another set of primers was used: SaNRAMP5(AANP)-pDR196F and SaNRAMP5-pDR196R. 1 μL of each PCR product was mixed and diluted 10-fold. Using this mixture as a PCR template, the SaNRAMP5(AANP) fragment was amplified using the SaNRAMP5-pDR196F / R primers and ligated into the pDR196 vector using the same method. Point mutations of other genes were constructed using a similar method.

[0027] Construction of the 35S:SaNRAMP5N-GFP vector:

[0028] Using a homologous recombination system, the 35S:SaNRAMP5N-GFP vector was constructed. The constructed SaNRAMP5-pDR196 plasmid was used as a template for PCR amplification and purification. The pCAMBIA1305 vector was digested with KpnI and SalI and purified. The primers for constructing the 35S:SaNRAMP5N-GFP vector were the same, only the template was different.

[0029] Rapid yeast conversion:

[0030] Pick a single colony of ycf1 from a YPDA plate and inoculate it into 4 ml of YPDA liquid medium. Incubate at 30°C and 225 rpm with shaking for 18-20 h (overnight) until OD600 = 0.8-1.0. Transfer 1 ml of the bacterial suspension to a 1.5 ml centrifuge tube and centrifuge at 4000 rpm for 5 min at room temperature. Discard the supernatant, resuspend the bacterial cells in 1 ml of sterile water, mix well, and centrifuge again to collect the bacteria. Discard the supernatant. Resuspend the bacterial cells in 1 ml of 0.1 M LiAc, mix well, mix well, and centrifuge again to collect the bacteria. Discard the supernatant. Finally, add 100 μl of 0.1 M LiAc to each 1.5 ml centrifuge tube for later use. Add the following reagents to each 1.5 ml centrifuge tube in sequence: 1 μg of target plasmid, 500 μl of 50% PEG3350Mix, and 5 μl of DTT. Then shake vigorously for about 1 min until completely mixed. Then, the 1.5 ml centrifuge tubes were incubated in a 30°C metal bath for 30 min, with vigorous vortexing every 10 min. Following this, a heat shock at 42°C for 20 min was performed, followed immediately by 5 min on ice, and then recovery at 30°C for 30 min. The cells were then collected by centrifugation at room temperature, 4000 rpm for 5 min, and the supernatant was discarded. For each transformation, the bacterial cells were resuspended in 200 μl of sterile water, gently mixed, and spread onto SD-U agar plates. After incubation at 30°C for 4 days, single clones were randomly selected for colony PCR verification.

[0031] Plant total protein extraction

[0032] Plant tissue (approximately 20 mg) was added to a mortar pre-cooled with liquid nitrogen and ground into a uniform powder. The powder was transferred to a 2.0 mL centrifuge tube, and 200–500 μL of total plant protein extract was added. The mixture was vortexed until completely dissolved, then placed on ice for 30 min, vortexing 1–2 times during this period. The tube was then centrifuged at 12,000 rpm for 10 min at 4 °C. The supernatant was carefully transferred to a new 1.5 mL EP tube, which can be used for subsequent purification of specific proteins or directly for Western blot analysis.

[0033] GFP protein purification

[0034] Pipettes of magnetic beads (Anti-GFP Nanobody MagaroseBeads) into 1.5 mL centrifuge tubes at a ratio of 1 mL magnetic beads to 300 mL extraction buffer. Place the tubes on a magnetic rack and wash twice with 1×TBST buffer (containing 1 mM PMSF). After separation, add the washed beads to the protein extraction buffer obtained in the previous step and incubate at 4°C for 1 hour. After incubation, separate the magnetic beads using a magnetic rack, discard the supernatant, add 1 mL of 1×TBST buffer, and wash the beads at 4°C for 10 minutes. Repeat this washing process three times. Transfer the magnetic beads to 1.5 mL centrifuge tubes, add 50 μL of 1×SDS loading buffer to resuspend the beads, heat the beads at 95°C for 10 minutes, and then separate the beads using a magnetic rack. Aspirate the supernatant to obtain the purified target protein.

[0035] Western blot analysis

[0036] After protein samples were separated by SDS-PAGE gel electrophoresis, they were transferred to PVDF membranes using a semi-dry transfer instrument (20V, 23min). After transfer, 5% BSA was added, and the membranes were blocked at room temperature (50rpm) for 1–2 hours or overnight at 4°C. Then, antibodies and primary antibody dilution buffers were mixed according to the required ratios for different antibodies. After blocking, primary antibody was added and incubated at 65rpm for 1–3 hours on a shaker. The primary antibody solution was then poured off, and the membranes were washed with 1×TBST for 10 minutes, repeated three times. Next, the corresponding secondary antibody dilution buffer was added, and the membranes were incubated at 65rpm for 45 minutes–1 hour on a shaker, followed by washing with 1×TBST for 10 minutes, repeated three times. Finally, ECL moon A solution and ECL moon B chemiluminescence solution were mixed in a 1:1 ratio. The membranes were removed, patted dry with absorbent paper, immersed in developing solution, and then exposed in an imaging system to observe the bands. The images were then saved.

[0037] Phosphorylation detection

[0038] The purified protein was run on an SDS-PAGE gel, transferred to a PVDF membrane, and incubated with 5% BSA at 65 rpm for 3-4 hours on a shaker. Then, it was incubated overnight with phosphorylation antibody working solution (Anti-Phosphoserine / threonine, PP2552). The dilution ratio of the phosphorylation antibody working solution was 1:1000, that is, 10 μL of Anti-Phosphoserine / threonine stock solution was diluted to 10 ml of 1×TBST solution. The diluted solution was the phosphorylation antibody working solution. After incubation, the appropriate secondary antibody (rabbit antibody) was selected and incubated at 65 rpm for 2 hours on a shaker. After incubation, the PVDF membrane was washed with 1×TBST for 5 minutes, and the washing was repeated 3 times. Finally, the phosphorylation results were obtained using ECL Moon developing solution and an imaging system.

[0039] Determination of cadmium or manganese content in yeast

[0040] Yeast cells in the logarithmic growth phase were transferred to liquid SD-U medium with an initial OD of 0.2 at 600 nm. The cells were cultured at 30°C and 200 rpm for 12 h, then 10 μM CdCl2 was added. The cells were cultured for another 2 h, then harvested by centrifugation at 4000 rpm and 4°C. The cells were washed three times with 10 μM EDTA (pH 8.0) at 4°C, then three times with ddH2O. The cells were lyophilized and digested in a microwave digester with 5 ml of analytical grade concentrated HNO3. The cadmium content was determined by ICP-MS.

[0041] Example 2: Comparison of Cadmium Absorption Capacity of Solanaceae Plants

[0042] Since black nightshade is genetically closely related to nightshade vegetables (such as potatoes, tomatoes, peppers, and eggplants), we transformed NRAMP5s from these species into the Δycf1 yeast strain and assessed their cadmium uptake capacity.

[0043] First, we performed yeast spot experiments to assess the susceptibility of yeast cells transformed with different NRAMP5s to cadmium. In the absence of Cd, no significant growth differences were observed between Δycf1 yeast cells containing the empty vector and yeast cells transformed with different NRAMP5s. However, on media containing different concentrations of Cd, yeast cells transformed with SaNRAMP5 exhibited the most pronounced Cd-susceptibility phenotype, followed by SmNRAMP5, StNRAMP5, SlNRAMP5, and CaNRAMP5. Figure 1 A). Yeast cadmium content determination showed that yeast cells transformed with SaNRAMP5 accumulated the highest Cd content, followed by SmNRAMP5, StNRAMP5, SlNRAMP5, and CaNRAMP5. Figure 1 B). Furthermore, we quantified the absolute expression levels of NRAMP5 in each plant species. The results showed that SaNRAMP5 expression in black nightshade was 4–5 times higher than that of StNRAMP5 and SlNRAMP5 in potato and tomato, respectively. Figure 1 C). To explain this difference, we investigated the cadmium uptake kinetics of yeast transformed with different NRAMP5s using CdCl2 concentrations ranging from 0 to 20 μM. The net Cd uptake in yeast cells transformed with each NRAMP5 was fitted to the Michaelis-Menten equation to determine the uptake kinetic parameters Vmax and Km. Figure 1 D). Subsequently, yeast growth curve experiments also confirmed this result. Figure 2 These data indicate that NRAMP5s exhibit significant differences in cadmium uptake capacity among different species.

[0044] Figure 1 In this study, (A) the pDR196 empty vector, SaNRAMP5, StNRAMP5, SlNRAMP5, CaNRAMP5, and SmNRAMP5 were transformed into the Δycf1 strain. Yeast cells containing different transformants were then spotted on SD-Ura medium containing different concentrations of CdCl2 for 3 days. (B) Cd content in the yeast Δycf1 strain transformed with pDR196 empty vector, SaNRAMP5, StNRAMP5, SlNRAMP5, CaNRAMP5, and SmNRAMP5. Data are mean ± SD, n = 5. Different letters indicate significant differences between different NRAMP5 transformants (P < 0.05, ANOVA). (C) Absolute expression levels of NRAMP5 in the roots of different Solanaceae species. Data are mean ± SD, n = 3. (D) Net Cd uptake in yeast was obtained by subtracting the Cd content in the pDR196 empty vector from the Cd content in each NRAMP5s transformant, and then the Michaelis-Menten equation was fitted to assess the differences in Cd uptake capacity of NRAMP5s from these species. Data are mean ± SD, n = 5.

[0045] Figure 2 The growth curves of Δycf1 yeast cells expressing the empty pDR196 vector, SaNRAMP5, StNRAMP5, SlNRAMP5, CaNRAMP5, and SmNRAMP5 are shown in the figure at specified time points under 0 and 8 μM CdCl2. Data are mean ± SD, n = 3.

[0046] In Cd-supplemented SD-U liquid medium, yeast cells expressing SaNRAMP5 showed the lowest OD values, followed by SmNRAMP5, StNRAMP5, SlNRAMP5, and CaNRAMP5. Figure 2 The Vmax of Cd uptake in yeast cells transformed with SaNRAMP5 was 21.0%, 45.7%, 124.9%, and 11.2% higher than that in yeast cells transformed with StNRAMP5, SlNRAMP5, CaNRAMP5, and SmNRAMP5, respectively. Figure 1 D), while the Km values ​​in yeast cells transformed with SaNRAMP5 were higher than those of other NRAMP5s.

[0047] Example 3: Comparison of NRAMP5 amino acid sequences in Solanaceae plants

[0048] SaNRAMP5, StNRAMP5, and SlNRAMP5 exhibit functional differences in cadmium uptake capacity, but their proteins share high sequence similarity, and protein evolutionary analysis shows that they cluster on a small branch. Figure 3Therefore, using total cDNA from the roots of tomatoes, potatoes, eggplants, and peppers as templates, we designed PCR primers to amplify the CDS sequences of SaNRAMP5, SmNRAMP5, StNRAMP5, SlNRAMP5, and CaNRAMP5, respectively. We then compared the amino acid sequences of NRAMP5 proteins from these five Solanaceae species. The comparison revealed that, compared to the other three species, StNRAMP5 and SlNRAMP5 proteins lacked four amino acids at their N-terminus: STNP( Figure 6 A). The amino acid sequence of Solanum nigrum SaNRAMP5 is shown in SEQ ID NO.1.

[0049] Example 4: Effects of STNP or AANP recovery in StNRAMP5 and SlNRAMP5

[0050] In this field, phosphorylation mutations are typically introduced using ST amino acids, while simulated dephosphorylation mutations are typically introduced using AA amino acids. This does not mean that dephosphorylation mutations can only use A amino acids to replace S or T amino acids. STNP amino acids were introduced into StNRAMP5 and SlNRAMP5 using overlap extension PCR and transformed into the Δycf1 yeast strain to assess their role in cadmium uptake. The results showed that STNP-filled StNRAMP5 and SlNRAMP5 significantly increased their sensitivity to Cd. Figure 4 AB). Furthermore, the determination of Cd content ( Figure 4 Both CD and growth curve measurements confirmed this. Figure 5 ).

[0051] exist Figure 4 In the study, (A) pDR196 empty vector, StNRAMP5, StNRAMP5+STNP, and StNRAMP5+AANP; (B) pDR196 empty vector, SlNRAMP5, SlNRAMP5+STNP, and SlNRAMP5+AANP. These transformed yeast cells were seeded on SD-Ura medium containing different concentrations of CdCl2 and cultured for 3–5 days. (C) Cadmium content in Δycf1 yeast strains transformed with pDR196 empty vector, StNRAMP5, StNRAMP5+STNP, and StNRAMP5+AANP. (D) Cadmium content in Δycf1 yeast strains transformed with pDR196 empty vector, SlNRAMP5, SlNRAMP5+STNP, and SlNRAMP5+AANP. Data are presented as mean ± standard deviation, n = 5. Different letters indicate significant differences in cadmium content between different NRAMP5 phosphorylation states and the pDR196 empty vector (P < 0.05, ANOVA).

[0052] exist Figure 5The growth curves of Δycf1 yeast cells expressing (A) pDR196 empty vector, SaNRAMP5, and SaNRAMP5(AANP), (B) pDR196 empty vector, StNRAMP5, StNRAMP5+STNP, and StNRAMP5+AANP, and (C) pDR196 empty vector, SlNRAMP5, SlNRAMP5+STNP, and SlNRAMP5+AANP were shown in the figure at 0 and 8 μM CdCl2. Data are presented as mean ± standard deviation, n = 3.

[0053] These results indicate the importance of STNP residues for the NRAMP5 protein in Cd uptake.

[0054] Example 5 simulates the effect of dephosphorylation mutation on the cadmium uptake capacity of SaNRAMP5.

[0055] Given that STNP residues are not located in the core domain or transmembrane region of the NRAMP5 protein, and that these residues contain potential serine (S) and threonine (T) phosphorylation sites, the inventors hypothesized that the stronger Cd uptake capacity of SaNRAMP5 might be due to phosphorylation at the N-terminus of the SaNRAMP5 protein. To verify this, the N-terminal region of SaNRAMP5 and SaNRAMP5(AANP) was expressed and purified from tobacco. Figure 6 B), the latter being a simulated non-phosphorylated form. Subsequently, a phosphorylation antibody was used to detect the purified protein. The results showed that a phosphorylated band was detected in the SaNRAMP5 protein, while no phosphorylated band was observed in the SaNRAMP5(AANP) protein. Figure 6 C) indicates that phosphorylation occurred at the N-terminus of SaNRAMP5, while no phosphorylation occurred after a non-phosphorylation mutation was introduced at the N-terminus of SaNRAMP5(AANP). Figure 6 The results showed that the introduction of a non-phosphorylation mutation significantly reduced the cadmium uptake capacity of SaNRAMP5 (AANP). Figure 6(A) Amino acid sequence alignment of CaNRAMP5, SaNRAMP5, SlNRAMP5, StNRAMP5, and SmNRAMP5. STNP residues are marked with red boxes. (B) Expression of the N-terminal regions of SaNRAMP5 and SaNRAMP5 (AANP) in tobacco via Agrobacterium-mediated infection. Proteins were purified using GFP antibody, and the purified proteins were visualized by Coomassie brilliant blue staining after gel running. (C) Phosphorylation signals were detected using anti-phosphoserine / threonine antibodies. (D) Δycf1 yeast cells transformed with pDR196 empty vector, SaNRAMP5, and SaNRAMP5 (AANP) were cultured on SD-Ura medium containing different concentrations of CdCl2 for 3–5 days. (E) Cadmium content in Δycf1 yeast strains transformed with pDR196 empty vector, SaNRAMP5, and SaNRAMP5 (AANP). Data are presented as mean ± standard deviation, n = 5. Different letters indicate significant differences between different forms of SaNRAMP5 and the empty pDR196 vector (P<0.05, ANOVA). STNP-mediated phosphorylation of the N-terminus of SaNRAMP5 protein leads to differences in its ability to absorb cadmium.

[0056] Example 6: The manganese absorption capacity of NRAMP5s is significantly different from that of cadmium, and the phosphorylation state of NRAMP5s does not affect manganese absorption.

[0057] In rice, OsNRAMP5 is the main transporter protein responsible for the absorption of the essential nutrient Mn. To investigate whether NRAMP5s in Solanaceae plants have Mn absorption function, we transformed NRAMP5s from these Solanaceae species into the Mn-deficient yeast strain Δsmf1 and spotted them on SD-U medium containing different concentrations of EGTA. The results showed that yeast cells transformed with StNRAMP5, SlNRAMP5, and SmNRAMP5 exhibited consistent and good growth on SD-U medium containing 25 mM. Figure 7 A). Surprisingly, yeast cells transformed with Δsmf1 and SaNRAMP5 showed weaker growth on 25 mM SD-U medium, while CaNRAMP5 showed the weakest growth phenotype. Figure 7 A). Mn content determination in yeast cells showed that the Mn content in yeast cells transformed with StNRAMP5, SlNRAMP5, and SmNRAMP5 was more than twice that in yeast cells transformed with SaNRAMP5. Figure 7 B). The yeast growth curve is consistent with the spot experiment and manganese content analysis. Figure 7 C). These studies indicate that NRAMP5s exhibit selective differences in their ability to absorb manganese and cadmium among different Solanaceae species. Figure 7In this study, (A) pDR196 empty vector, SaNRAMP5, StNRAMP5, SlNRAMP5, CaNRAMP5, and SmNRAMP5 were transformed into Δsmf1 cells. The yeast cells were then spotted on SD-Ura medium containing different concentrations of EGTA for 3–5 days. (B) Mn content in yeast Δsmf1 cells transformed with pDR196 empty vector, SaNRAMP5, StNRAMP5, SlNRAMP5, CaNRAMP5, and SmNRAMP5. Data are mean ± SD, n = 5. Different letters indicate significant differences between each transformant (P < 0.05, ANOVA). (C) Growth curves of Δsmf1 yeast cells expressing pDR196 empty vector, SaNRAMP5, StNRAMP5, SlNRAMP5, CaNRAMP5, and SmNRAMP5 at different time points under 0 and 20 mM EGTA. Data are mean ± SD, n = 3.

[0058] Previous studies have confirmed the effect of NRAMP5s phosphorylation on cadmium absorption. The inventors further investigated the effect of NRAMP5s phosphorylation status on its manganese absorption capacity. Interestingly, yeast culture results showed that NRAMP5s supplemented with either STNP or AANP did not exhibit significantly different growth rates compared to their original protein on SD-U medium containing 25 mM. This indicates that the phosphorylation status of NRAMP5s has no effect on manganese absorption. Figure 8 AC). Subsequently, we measured the Mn content in yeast cells and found no difference in Mn content between non-phosphorylated and converted yeast cells transformed with SaNRAMP5, StNRAMP5, and SlNRAMP5. Figure 8 These results indicate that the phosphorylation state of NRAMP5s does not affect their uptake of Mn.

[0059] exist Figure 8In the following steps: (A) pDR196 empty vector, SaNRAMP5, and SaNRAMP5 (AANP) were transformed into Δsmf1 cells. The yeast cells were then plated on SD-Ura medium containing different concentrations of EGTA for 3-5 days. (B) Δsmf1 yeast cells transformed with pDR196 empty vector, SaNRAMP5, SaNRAMP5+STNP, and SaNRAMP5+AANP were plated on SD-Ura medium containing different concentrations of EGTA for 3-5 days. (C) Δsmf1 yeast cells transformed with pDR196 empty vector, SaNRAMP5, SaNRAMP5+STNP, and SaNRAMP5+AANP were plated on SD-Ura medium containing different concentrations of EGTA for 3-5 days. Transformation (D) of pDR196 empty vector, SaNRAMP5, and SaNRAMP5(AANP); (E) of pDR196 empty vector, StNRAMP5, StNRAMP5+STNP, and StNRAMP5+AANP; (F) Mn content in yeast strain Δsmf1 transformed with pDR196 empty vector, SlNRAMP5, SlNRAMP5+STNP, and SlNRAMP5+AANP. Data are mean ± SD, n = 5. Different letters indicate significant differences in manganese content between phosphorylated forms of NRAMP5s and pDR196 empty vector (P < 0.05, ANOVA).

[0060] Identifying the substrate selectivity of plant transporters is crucial for balancing plant growth and minimizing the accumulation of harmful metals. STNP-mediated phosphorylation affects the selective uptake of cadmium by NRAMP5 in plants. Mutations mimicking non-phosphorylation reduce cadmium uptake in plants but do not affect manganese uptake.

[0061] Obviously, those skilled in the art can make various modifications and variations to this application without departing from the spirit and scope of this application. Therefore, if such modifications and variations fall within the scope of the claims of this application and their equivalents, this application also intends to include such modifications and variations.

Claims

1. A method for reducing cadmium absorption from solanum, characterized in that, Dephosphorylation was performed on amino acids 27-28 at the N-terminus of the NRAMP5 protein of Solanum nigrum, with SEQ ID NO.1 as the reference sequence.

2. The method as described in claim 1, characterized in that, The dephosphorylation treatment involves mutating the amino acid to alanine.

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