Method for regulating and controlling selective absorption of cadmium and manganese by plants through dephosphorylation

Abstract

The invention relates to a method for regulating and controlling selective absorption of cadmium and manganese by plants through dephosphorylation. The STNP-mediated phosphorylation is crucial to the selective uptake of cadmium and manganese of solanaceae NRAMP5 (Nuclear Receptor Amplification Protein 5). Simulation of non-phosphorylated mutation in NRAMP5 reduces the absorption of plants to cadmium without affecting the absorption of plants to manganese.

Description

Technical Field

[0001] The present application belongs to the field of genetic engineering technology, and specifically relates to a method for regulating the selective absorption of cadmium and manganese by plants through dephosphorylation. Background Art

[0002] Cadmium (Cd) pollution in the environment poses a major threat to food safety and human health due to its high toxicity and its easy uptake into the food chain through plant roots. Researchers have been able to reduce Cd accumulation by identifying genes responsible for Cd accumulation in plants and knocking out these genes. However, while these genes promote Cd uptake, they may also play a role in the uptake of essential nutrients such as manganese. For example, knocking out OsNRAMP5 in rice significantly reduced Cd accumulation in rice, but it also caused a decrease in Mn accumulation in rice, resulting in losses in rice growth and yield. Therefore, a dilemma is created between reducing Cd uptake and stabilizing the need for essential metals. Summary of the invention

[0003] In order to solve the above technical problems, the present application provides a method for reducing cadmium absorption in plants, comprising dephosphorylating the N-terminus of the NRAMP5 protein of the plant.

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

[0005] In some embodiments, the dephosphorylation treatment is directed to amino acids 27-28 or 27-30 with SEQ ID NO. 1 as the reference sequence.

[0006] In some embodiments, the dephosphorylation treatment targets two consecutive amino acids of ST or four consecutive amino acids of STNP at the N-terminus of the plant NRAMP5 protein.

[0007] In some embodiments, the dephosphorylation treatment is to perform non-phosphorylation mutation on 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, the dephosphorylation treatment is to delete two consecutive amino acids of ST or four consecutive amino acids of STNP at the N-terminus of the plant NRAMP5 protein.

[0009] The present application also provides a method for increasing cadmium absorption in plants, comprising site-specifically complementing 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 embodiments, the position of the site-directed complementation is immediately after the 26th amino acid in the reference sequence of SEQ ID NO.1.

[0011] In some embodiments, the position of the site-specific complementation is immediately after three consecutive amino acids of the N-terminal LPS of the NRAMP5 protein of the plant.

[0012] The present application also provides a method for predicting the cadmium absorption capacity of plants, comprising 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 so, it is predicted that the plant has a strong cadmium absorption capacity; if not, it is predicted that the plant has a weak cadmium absorption capacity.

[0013] In some embodiments, the plant is a monocot or a dicot, preferably, the plant is a Solanaceae plant, such as one or more of Solanum nigrum, tomato, potato, eggplant or pepper.

[0014] The beneficial effects of the present application are as follows: by discovering and utilizing the substrate selectivity of NRAMP5, the accumulation of harmful metals (such as cadmium) is minimized, while not affecting the acquisition of essential metal elements (such as manganese) for plant growth. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 : Identification of cadmium uptake capacity of SaNRAMP5, StNRAMP5, S l NRAMP5, CaNRAMP5 and SmNRAMP5.

[0016] Figure 2 : Growth curves of Δycf1 yeast cells transformed with different NRAMP5s at 0 and 8 μM CdCl21.

[0017] Figure 3 : Phylogenetic tree of NRAMP5 proteins.

[0018] Figure 4 : Effects of replenishing STNP or AANP residues in StNRAMP5 and S l NRAMP5 on their cadmium uptake capacity.

[0019] Figure 5 : Growth curves of different Δycf1 yeast cells transformed with different NRAMP5s at 0 and 8 μM CdCl22.

[0020] Figure 6 : Effect of dephosphorylation of SaNRAMP5 on cadmium uptake capacity.

[0021] Figure 7 : Characterization of Mn uptake capacity of SaNRAMP5, StNRAMP5, S l NRAMP5, CaNRAMP5 and SmNRAMP5.

[0022] Figure 8 : Characterization of the effect of the phosphorylation status of NRAMP5s on manganese uptake capacity. DETAILED DESCRIPTION

[0023] In order to specifically illustrate the universal design concept of the present application, specific experimental parameters are used as an example below, but this should not be used as a reason to limit the scope of protection of the present application.

[0024] The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples are all commercially available unless otherwise specified.

[0025] Example 1 Experimental design ideas and methods

[0026] Using the total cDNA of tomato, potato, eggplant and pepper root as template, 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 3min, 95℃ denaturation for 30s, 56℃ annealing and 72℃ annealing extension for 1min 20s, 35 cycles, 72℃ 7min, the amplified PCR products were detected by 1% agarose gel electrophoresis, and the fragment size was about 1600bp. The target PCR products were separated by agarose electrophoresis and then cut and recovered. The yeast expression vector pDR196 was double-digested with EcoR1 and Xho1 and purified and recovered. The linearized plasmid and the purified PCR product of each gene were subjected to homologous recombination reaction: the system was 5 μL, including 1 μL linearized vector and 4 μL PCR purified product, and then the reaction was carried out at 37 degrees for 1 hour for transformation, and a single clone was picked for sequencing. The correct single clone was preserved, and the plasmid was named StNRAMP5-pDR196, SlNRAMP5-pDR196, SmNRAMP5-pDR196 and CaNRAMP5-pDR196. The construction of point mutations was performed using the corresponding constructed vectors connected to pDR196 as templates for overlap extension PCR. Specifically, SaNRAMP5-pDR196F and SaNRAMP5(AANP)-pDR196R were used as a set of primers, and SaNRAMP5(AANP)-pDR196F and SaNRAMP5-pDR196R were used as a set of primers. 1 μL of each of the obtained PCR products was mixed and diluted 10 times. This was used as a PCR template and SaNRAMP5-pDR196F / R primers were used for amplification to obtain the fragment of SaNRAMP5(AANP), which was then connected to the pDR196 vector using the same method as above. Similar methods were used for point mutations of other genes.

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

[0028] The homologous recombination system was used to construct the 35S:SaNRAMP5N-GFP vector. The constructed SaNRAMP5-pDR196 plasmid was used as a template for PCR amplification and purification. The pCAMBIA1305 vector was double-digested with KpnI and SalI and purified. The primers for constructing the 35S:SaNRAMP5(AANP)N-GFP vector were the same, but the templates were different.

[0029] Yeast rapid transformation:

[0030] Pick a single colony of ycf1 from the YPDA plate and inoculate it into 4ml of YPDA liquid medium, 30℃, 225rpm, shake and culture for 18-20h (overnight) until OD600 = 0.8-1.0. Take 1ml of bacterial solution to a 1.5ml centrifuge tube, room temperature, 4000rpm, 5min, discard the supernatant, resuspend the bacteria with 1ml sterile water, blow evenly, centrifuge again to collect the bacteria, and discard the supernatant. Resuspend the bacteria with 1ml 0.1M LiAc, mix, blow evenly, centrifuge again to collect the bacteria, and discard the supernatant. Finally, add 100μl 0.1M LiAc to each 1.5ml centrifuge tube to resuspend the bacteria for use. Add the following reagents to each 1.5ml centrifuge tube in sequence: 1μg target plasmid, 500μl 50% PEG3350Mix, 5μl DTT, and then shake vigorously for about 1min to completely mix. Then place the 1.5ml centrifuge tube in a 30℃ metal bath and incubate for 30min, vortexing vigorously every 10min. Then heat shock at 42℃ for 20min, immediately place on ice for 5min, and then recover at 30℃ for 30min. Centrifuge to collect bacteria, at room temperature, 4000rpm, 5min, discard the supernatant. Suspend the bacteria with 200μl sterile water for each transformation, mix as gently as possible, apply to SD-U solid plate, and culture at 30℃ for 4 days, then randomly pick single clones for colony PCR verification.

[0031] Plant total protein extraction

[0032] Add plant tissue (about 20 mg) to a mortar pre-cooled with liquid nitrogen and grind into a uniform powder. Use a medicine spoon to scoop into a 2.0 mL centrifuge tube, add 200-500 μL of plant total protein extract, vortex to mix, and place on ice for 30 minutes after thorough dissolution. During this period, use a vortex instrument to mix 1-2 times, and then centrifuge at 4°C, 12,000 rpm for 10 minutes. Carefully transfer the supernatant to a new 1.5 mL EP tube, which can be used for subsequent purification of specific proteins or directly for Western blot.

[0033] GFP protein purification

[0034] Magnetic beads (Anti-GFP Nanobody Magarose Beads) were aspirated into a 1.5 ml centrifuge tube at a ratio of 1 mL magnetic beads / 300 mL extract, and placed on a magnetic rack. The magnetic beads were washed twice with 1×TBST (containing 1 mM PMSF) buffer, and after separation, they were added to the protein extract obtained in the previous step, and incubated at 4°C for 1 h. After incubation, the magnetic beads were separated with a magnetic rack, the supernatant was discarded, 1 mL 1×TBST buffer was added, and the magnetic beads were washed with rotation at 4°C for 10 min, and the washing was repeated three times. The magnetic beads were transferred to a 1.5 mL centrifuge tube, 50 ul 1×SDS loading buffer was added to resuspend the magnetic beads, and the magnetic beads were heated at 95°C for 10 minutes. The magnetic beads were separated by standing on a magnetic rack, and the supernatant was aspirated to obtain the purified target protein.

[0035] Western blot detection

[0036] After the protein sample was separated by SDS-PAGE gel electrophoresis, the protein was transferred to a PVDF membrane (20V, 23min) using a semi-dry transfer instrument. After the transfer, 5% BSA was added and blocked at room temperature at 50rpm for 1 to 2h or at 4°C overnight; then the antibody and primary antibody diluent were mixed according to the required ratio of different antibodies, and the primary antibody was added for incubation after the blocking, and the shaking was incubated at 65rpm for 1h to 3h. After pouring out the primary antibody solution, 1×TBST was added for washing for 10min, and repeated three times. Then the corresponding secondary antibody diluent was added, incubated at 65rpm for 45min to 1h, and then washed with 1×TBST for 10min, and repeated three times; finally, ECL moon A solution and ECL moon B luminescent solution were mixed in a 1:1 ratio, the membrane was taken out and wiped with absorbent paper to remove excess water, and then immersed in the developer, and then placed in the imager for exposure to observe the strips, and the imaging photos were saved.

[0037] Phosphorylation detection

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

[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 initial OD 600nm = 0.2, cultured at 30℃ and 200rpm for 12h, 10μM CdCl2 was added, and the culture was continued for 2h. Yeast cells were harvested by centrifugation at 4000rpm at 4℃, washed three times with 10μΜ EDTA (pH 8.0) at 4℃, then washed three times with ddH2O, freeze-dried, digested with 5ml of high-grade pure concentrated HNO3 in a microwave digester, and the cadmium content was determined by ICP-MS.

[0041] Example 2 Comparison of the ability of Solanaceae plants to absorb cadmium

[0042] Because Solanum nigrum is genetically closely related to Solanaceae vegetables (e.g., potato, tomato, pepper, and eggplant), we transformed NRAMP5s from each of these species into the Δycf1 yeast strain and evaluated their Cd uptake capacity.

[0043] First, we performed yeast dot assays to evaluate the sensitivity of yeast cells transformed with different NRAMP5s to Cd. In the absence of Cd, no significant growth differences were observed between Δycf1 yeast cells containing an empty vector and yeast cells transformed with different NRAMP5s. However, on medium containing different concentrations of Cd, yeast cells transformed with SaNRAMP5 exhibited the most obvious Cd-sensitive phenotype, followed by SmNRAMP5, StNRAMP5, SlNRAMP5, and CaNRAMP5 ( Figure 1 A). Yeast cadmium content assay showed that yeast cells transformed with SaNRAMP5 accumulated the highest Cd content, followed by SmNRAMP5, StNRAMP5, SlNRAMP5, and CaNRAMP5 ( Figure 1 B). In addition, we quantified the absolute expression level of NRAMP5 in each plant. The results showed that the expression of SaNRAMP5 in Solanum nigrum was 4-5 times that of StNRAMP5 and SlNRAMP5 in potato and tomato, respectively ( Figure 1 C). To explain this discrepancy, 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 NRAMP5s 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 suggest that NRAMP5s exhibit significant differences in Cd uptake capacity among different species.

[0044] Figure 1 (A) pDR196 empty vector, SaNRAMP5, StNRAMP5, SlNRAMP5, CaNRAMP5 and SmNRAMP5 were transformed into Δycf1 strain, respectively. Then the yeast cells containing the above different transformants were spotted on SD-Ura medium containing different concentrations of CdCl2 for 3 days. (B) Cd content in 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 NRAMP5s transformants (P < 0.05, ANOVA). (C) Absolute expression level of NRAMP5 in roots of different Solanaceae species. Data are mean ± SD, n = 3. (D) The 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 fitting the Michaelis-Menten equation to evaluate the differences in Cd uptake capacity of NRAMP5s from these species. Data are mean ± SD, n = 5.

[0045] Figure 2 Growth curves of Δycf1 yeast cells expressing pDR196 empty vector, SaNRAMP5, StNRAMP5, SlNRAMP5, CaNRAMP5, and SmNRAMP5 at 0 and 8 μM CdCl2 for the indicated times are shown in Figure 2. Data are mean ± SD, n = 3.

[0046] In SD-U liquid medium supplemented with Cd, the growth of yeast cells expressing SaNRAMP5 showed the lowest OD value, 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 amino acid sequences of NRAMP5 from Solanaceae plants

[0048] SaNRAMP5, StNRAMP5 and SlNRAMP5 have functional differences in cadmium absorption capacity, but their proteins have high sequence similarity, and protein evolution analysis shows that they are clustered in a small branch ( Figure 3Therefore, we used the total cDNA of tomato, potato, eggplant and pepper roots as templates to design PCR primers to amplify the CDS sequences of SaNRAMP5, SmNRAMP5, StNRAMP5, SlNRAMP5 and CaNRAMP5, respectively, and compared the amino acid sequences of NRAMP5 proteins of the five Solanaceae species. The comparison results showed that compared with the other three species, StNRAMP5 and SlNRAMP5 proteins lacked four amino acids at the 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 replenishing STNP or AANP in StNRAMP5 and SlNRAMP5

[0050] In the art, ST amino acids are usually used to introduce phosphorylation mutations, while AA amino acids are usually used to simulate dephosphorylation mutations. 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 evaluate their role in cadmium absorption. The results showed that StNRAMP5 and SlNRAMP5 supplemented with STNP significantly increased their sensitivity to Cd ( Figure 4 AB). Moreover, the determination of Cd content ( Figure 4 This was confirmed by CD) and growth curve assays ( Figure 5 ).

[0051] exist Figure 4 (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 inoculated on SD-Ura medium containing different concentrations of CdCl2 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 mean ± SD, n = 5. Different letters indicate significant differences in cadmium content between different NRAMP5 phosphorylation states and pDR196 empty vector (P < 0.05, ANOVA).

[0052] exist Figure 5Growth curves of Δycf1 yeast cells expressing (A) pDR196 empty vector, SaNRAMP5 and SaNRAMP5(AANP), (B) pDR196 empty vector, StNRAMP5, StNRAMP5+STNP and StNRAMP5+AANP, (C) pDR196 empty vector, SlNRAMP5, SlNRAMP5+STNP and SlNRAMP5+AANP under 0 and 8 μM CdCl2. Data are mean ± SD, n = 3.

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

[0054] Example 5 Simulation of the effect of dephosphorylation mutation on the cadmium absorption capacity of SaNRAMP5

[0055] Considering that the STNP residues are not 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 speculated that the stronger Cd absorption capacity of SaNRAMP5 may be caused by the phosphorylation of the N-terminus of the SaNRAMP5 protein. To verify this, the N-terminal regions of SaNRAMP5 and SaNRAMP5(AANP) were expressed and purified from tobacco ( Figure 6 B), the latter is a simulated non-phosphorylated form. Subsequently, the purified protein was detected using a phosphorylation antibody. The results showed that phosphorylation bands were detected in the SaNRAMP5 protein, while no phosphorylation bands were observed in the SaNRAMP5(AANP) protein ( Figure 6 C), namely, phosphorylation occurred at the N-terminus of SaNRAMP5, whereas no phosphorylation occurred after a non-phosphorylation mutation was introduced into the N-terminus of SaNRAMP5(AANP). Figure 6 The results showed that the cadmium absorption capacity of SaNRAMP5 (AANP) was significantly reduced after the introduction of non-phosphorylation mutations. Figure 6(A) Amino acid sequence alignment of CaNRAMP5, SaNRAMP5, SlNRAMP5, StNRAMP5 and SmNRAMP5. The STNP residues are marked with red boxes. (B) The N-terminal regions of SaNRAMP5 and SaNRAMP5(AANP) were expressed in tobacco by Agrobacterium-mediated infection. The proteins were purified using GFP antibodies, and the purified proteins were run on gels and visualized by Coomassie Brilliant Blue staining. (C) Phosphorylation signals were detected using anti-phosphoserine / threonine antibodies. (D) Δycf1 yeast cells transformed with pDR196 empty vector, SaNRAMP5 and SaNRAMP5(AANP) were dotted and 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 mean ± SD, n = 5. Different letters indicate significant differences between different forms of SaNRAMP5 and pDR196 empty vector (P<0.05, ANOVA). STNP-mediated phosphorylation at the N-terminus of SaNRAMP5 protein leads to different effects on its cadmium absorption capacity.

[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 responsible for absorbing the essential nutrient element Mn. To investigate whether NRAMP5s in Solanaceae plants have the function of absorbing Mn, we transformed the NRAMP5s of these Solanaceae species into the Mn absorption-deficient yeast strain Δsmf1 and spotted it on SD-U medium containing different concentrations of EGTA. The results showed that yeast cells transformed with StNRAMP5, SlNRAMP5, and SmNRAMP5 Δsmf1 grew uniformly and well on SD-U medium containing 25mM EGTA ( Figure 7 A). Unexpectedly, yeast cells transformed with SaNRAMP5 and Δsmf1 showed weak growth in the medium containing 25 mM SD-U, while CaNRAMP5 showed the weakest growth phenotype ( Figure 7 A). The Mn content in yeast cells was determined to be more than twice that in yeast cells transformed with StNRAMP5, SlNRAMP5, and SmNRAMP5 ( Figure 7 B). The yeast growth curve is consistent with the dot experiment and manganese content analysis ( Figure 7 C) These studies suggest that there are selective differences in the uptake capacity of NRAMP5s for manganese and cadmium among different Solanaceae species. Figure 7In the figure, (A) pDR196 empty vector, SaNRAMP5, StNRAMP5, SlNRAMP5, CaNRAMP5 and SmNRAMP5 were transformed into Δsmf1, respectively. Then the yeast cells were 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 times 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 studied the effect of NRAMP5s phosphorylation status on its manganese absorption capacity. Interestingly, the yeast dot results showed that there was no significant difference in the growth of NRAMP5s supplemented with STNP or AANP compared with its original protein in the medium containing 25mM SD-U, indicating that the phosphorylation status of NRAMP5s has no effect on manganese absorption ( Figure 8 AC). We then measured the Mn content in yeast cells and found that there was no difference in the Mn content between yeast cells transformed with the non-phosphorylated and native proteins of SaNRAMP5, StNRAMP5, and SlNRAMP5 ( Figure 8 DF). These findings suggest that the phosphorylation status of NRAMP5s does not affect its Mn uptake.

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

[0060] Identifying the substrate selectivity of plant transporters is essential for balancing plant growth and minimizing the accumulation of deleterious metals. STNP-mediated phosphorylation affects the selective uptake of cadmium by plant NRAMP5. A non-phosphorylating mutation reduces cadmium uptake in plants but does not affect manganese uptake.

[0061] Obviously, those skilled in the art can make various changes and modifications to the present application without departing from the spirit and scope of the present application. Thus, if these modifications and variations of the present application fall within the scope of the claims of the present application and their equivalents, the present application is also intended to include these modifications and variations.

Claims

1. A method for reducing cadmium absorption in plants, characterized in that: The N-terminus of the NRAMP5 protein of the plant is dephosphorylated.

2. The method according to claim 1, characterized in that The N-terminus of the plant NRAMP5 protein is the first 41 amino acids at the N-terminus with SEQ ID NO.1 as the reference sequence, preferably the first 30, 31, 32, 33, 34, or 35 amino acids at the N-terminus.

3. The method according to claim 1 or 2, characterized in that The dephosphorylation treatment is directed to amino acids 27-28 or 27-30 based on SEQ ID NO. 1 as a reference sequence.

4. The method according to any one of claims 1 to 3, characterized in that: The dephosphorylation treatment targets two consecutive amino acids of ST or four consecutive amino acids of STNP at the N-terminus of the plant NRAMP5 protein.

5. The method according to any one of claims 1 to 4, characterized in that: The dephosphorylation treatment is to perform non-phosphorylation mutation on two consecutive amino acids of ST or four consecutive amino acids of STNP at the N-terminus of the plant NRAMP5 protein.

6. The method according to any one of claims 1 to 5, characterized in that: The dephosphorylation treatment is to delete two consecutive amino acids of ST or four consecutive amino acids of STNP at the N-terminus of the plant NRAMP5 protein.

7. A method for increasing cadmium absorption in plants, characterized in that: Two consecutive amino acids of ST or four consecutive amino acids of STNP are site-specifically complemented into the N-terminus of the NRAMP5 protein of the plant, and 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.

8. The method according to claim 7, characterized in that The position of the site-directed complementation is immediately after the 26th amino acid in the reference sequence of SEQ ID NO.

1.

9. The method according to claim 7 or 8, characterized in that The position of the site-specific complementation is immediately after three consecutive amino acids of the N-terminal LPS of the NRAMP5 protein of the plant.

10. A method for predicting the cadmium absorption capacity of plants, characterized in that: The method comprises the steps of detecting whether the N-terminus of the NRAMP5 protein of the plant has two consecutive ST amino acids or four consecutive STNP amino acids; if so, it is predicted that the plant has a strong ability to absorb cadmium; if not, it is predicted that the plant has a weak ability to absorb cadmium.

Images