Method for reducing cadmium accumulation in rice by using osnramp4 gene

Abstract

The application discloses a method for reducing plant cadmium accumulation by using Nramp aluminum ion transporter NRAMP4, and comprises the following steps: down-regulating, inactivating, weakening the function of or knocking out the Nramp aluminum ion transporter NRAMP4 gene expression in the chromosome of a wild type plant, so that the cadmium absorption and accumulation of the plant, such as rice roots and aboveground parts, can be significantly reduced. The application provides a genetic resource for cultivating a low-cadmium accumulation plant variety.

Description

Technical Field

[0001] This invention belongs to the field of agricultural biotechnology and relates to a method for reducing cadmium accumulation in rice using the OsNRAMP4 gene. Specifically, it relates to a method for reducing cadmium accumulation in rice by downregulating the expression of the OsNRAMP4 gene, a natural resistance-related macrophage aluminum ion transporter. Background Technology

[0002] Cadmium pollution in agricultural soils and food crops has become a major challenge threatening the ecological environment and food safety. According to the "National Soil Pollution Status Survey Bulletin" (2014) jointly released by the Ministry of Environmental Protection and the Ministry of Land and Resources, cadmium ranked first among heavy metal pollutants with an exceedance rate of 7.0%, becoming a core pollutant threatening arable land safety. Cadmium's harmful effects on human health include chronic kidney damage, skeletal system destruction, carcinogenicity, and multi-system toxicity.

[0003] Cadmium ingestion through food is the primary cause of cadmium harm, and rice has an extremely strong capacity to accumulate cadmium, making it a major factor in cadmium intake through food. Itai-itai disease in Japan is a result of long-term consumption of cadmium-contaminated rice, leading to kidney damage and abnormal bone metabolism. Currently, reducing cadmium content in rice through genetic regulation, cultivation techniques, and phytoremediation strategies is an effective means of addressing cadmium accumulation in rice.

[0004] Many mineral elements are first absorbed into the plant root system through relatively specific transport proteins, and then transported to the above-ground parts and seeds through other transport proteins. Since cadmium (Cd) is not an essential element for plant growth, plants mainly transport divalent Cd through divalent metal ion transporters such as manganese (Mn), iron (Fe), or zinc (Zn) ions. 2+ Ions. The root is the main site where plants absorb cadmium from the soil, and the xylem plays a key role in transporting cadmium from the roots to the above-ground parts. This step is a key factor in determining the accumulation of cadmium in plants.

[0005] In studies on cadmium uptake and distribution in rice, several transporter protein families have been identified in rice roots involved in cadmium transport, such as the natural resistance-associated macrophage protein (NRAMP or Nramp) family, the heavy metal transporting ATPase (HMA) family, and the zinc-regulated transporters and iron-regulated transporter-like protein (ZIP) family. Among these, the NRAMP family is generally considered to be the most active transporter, playing a crucial role in cadmium uptake by roots. OsNRAMP5 is the main transporter protein for cadmium uptake in rice roots, primarily responsible for manganese (Mn) uptake, but due to its non-specificity to divalent metal ions, it also mediates cadmium uptake (Sasaki et al., 2012). Other members, such as OsNRAMP1, are induced to express under iron (Fe) deficiency conditions and mainly participate in manganese uptake, but also non-specifically transport cadmium (Chang et al., 2020). OsNRAMP2 is located on the vacuolar membrane and promotes the transport of cadmium from the roots to the aboveground parts by positively regulating the efflux of cadmium from vacuoles (Yang et al., 2023). OsNRAMP3 is a manganese transporter located in the vascular bundle and is involved in the reuse of manganese from old leaves to new leaves. Other members of this family, OsNRAMP4, also known as OsNRAT1, has been reported as a trivalent aluminum ion transporter located on the plasma membrane (Xia et al., 2010), but its ability to transport divalent metal ions such as manganese or cadmium ions in rice is still unclear.

[0006] Since rice is a staple food in many regions, the accumulation of cadmium in rice poses a serious threat to public health. Therefore, there is an urgent need in this field to develop a method to reduce the cadmium content in rice. Summary of the Invention

[0007] Our research group has conducted systematic studies on reducing cadmium accumulation in rice. For example, patent document CN202411390336.3 reported that the histone deacetylase OsHDA703 (NCBI accession number BAS77624.1) has a negative regulatory effect on cadmium accumulation in rice. In further research and screening of proteins and genes related to cadmium accumulation in rice, we discovered a new mechanism that regulates the accumulation of trivalent aluminum ions (Al) in rice. 3+The Nramp (Natural Resistance-Associated Macrophage Protein, NRAMP) aluminum ion transporter 1, OsNRAMP4 (also noted as OsNRAT1, NCBI accession number AB525887), is involved in transmembrane transport of divalent cadmium ions (Cd). 2+ It also has a significant regulatory effect on transmembrane transport, but for divalent manganese ions Mn 2+ The regulatory effect of OsNRAMP4 on transmembrane transport is relatively small, exhibiting an imbalance and specificity / selectivity in the regulation of transmembrane transport of divalent metal ions. In the japonica rice Nipponbare (NPB) or indica rice Kasalath (Kasa) varieties, loss of function of the OsNRAMP4 gene, or knockout using CRISPR / Cas9, significantly reduced cadmium ion absorption and accumulation in the roots and aboveground parts of rice. However, it had almost no effect on manganese ion absorption and accumulation, suggesting that the OsNRAMP4 gene could be used as a target for breeding low-cadmium rice varieties. Specifically, this invention provides the following technical solution.

[0008] The first aspect of the invention provides the use of Nramp aluminum ion transporter (abbreviated NRAMP4) for reducing cadmium accumulation in plants.

[0009] In one embodiment, the above-mentioned use is a method for reducing cadmium accumulation in plants by targeting the Nramp aluminum ion transporter, comprising the following steps: downregulating, inactivating, weakening or knocking out the Nramp aluminum ion transporter gene NRAMP4 in the chromosomes of wild-type plants, resulting in a reduction in the absorption and storage of heavy metal cadmium (Cd) in the roots of plants such as rice and / or a reduction in the translocation of cadmium from the roots to the aboveground parts.

[0010] Optionally, the above-mentioned Nramp aluminum ion transporter is the rice-derived Nramp aluminum ion transporter OsNRAMP4 (also annotated as OsNRAT1, NCBI accession number AB525887, amino acid sequence SEQ ID NO:2), and the nucleotide sequence of the coding region of its expression gene OsNRAMP4, namely CDS, is SEQ ID NO:1.

[0011] The above can be implemented in the following ways:

[0012] (1) Knock out the Nramp aluminum ion transporter gene NRAMP4 in the chromosomes of wild-type plants;

[0013] (2) Downregulate the expression level of the gene NRAMP4 in the chromosomes of wild-type plants;

[0014] (3) Replace the NRAMP4 gene in the chromosomes of wild-type plants with a mutant of the NRAMP4 gene that has lost or downregulated function; and / or

[0015] (4) Block, inhibit or interfere with the expression of the gene NRAMP4 in the chromosomes of wild-type plants.

[0016] Furthermore, the above method (2) can be selected from the following group:

[0017] (2-1) Mutations in the promoter region and / or coding region of the NRAMP4 gene lead to downregulation of the expression level of the NRAMP4 gene;

[0018] (2-2) Mutations in the upstream regulators of the NRAMP4 gene lead to a downregulation of NRAMP4 expression; or

[0019] (2-3) Introduce interacting proteins of Nramp aluminum ion transporter into wild-type plants to alter the function of the NRAMP4 gene.

[0020] For example, the mutation in the coding region described in method (2-1) is a frameshift mutation, which leads to the inactivation or loss of function of the Nramp aluminum ion transporter; or it is the insertion of a retrotransposon in the coding region, which causes the protein to be translated incorrectly and cannot encode the correct NRAMP4 protein.

[0021] When the plant is rice, the mutation in the coding region described in method (2-1) can be a frameshift mutation, such as a two-base (AA) deletion mutation at positions 113 and 114 in the coding region of the gene OsNRAMP4 (NCBI accession number AB525887), i.e., the CDS nucleotide sequence SEQ ID NO:1, namely the OsNRAMP4-AA mutation, which causes premature termination of translation of the OsNRAMP4 protein, referred to in this paper as the nramp4-2 mutant; or

[0022] The mutation in the coding region described in method (2-1) is the insertion of the tos-17 retrotransposon into the coding region of the gene OsNRAMP4 (NCBI accession number AB525887), i.e., the CDS nucleotide sequence SEQ ID NO:1 (reported in the literature Xia, J., Yamaji, N., Kasai, T., & Ma, JF (2010). Plasma membrane-localized transporter for aluminuminrice. Proceedings of the National Academy of Sciences, 107(43), 18381-18385.), which is referred to as the nramp4-1 mutant in this paper.

[0023] The steps described above can be implemented using gene editing technology, antisense nucleic acids, and transcriptional regulation.

[0024] The gene editing technology may be selected from the following group: homologous double crossover, TALEN system, CRISPR-Cas9 system, CRISPR-Cpf1 system, CRISPR-Cas12 system, CRISPR-BEST system, and MuGENT.

[0025] Preferably, the plant is a crop selected from the group consisting of rice, wheat, corn, soybean, barley, oats, rye, sorghum, cotton, vegetables, tobacco, and cruciferous plants. Preferably, the plant is rice, and the rice is japonica or indica rice.

[0026] In one specific implementation, the above-mentioned use is to target the Nramp aluminum ion transporter gene to cultivate plant varieties with low cadmium accumulation.

[0027] This invention discovers a novel method for regulating trivalent aluminum ions (Al) in rice. 3+ The transmembrane aluminum ion transporter OsNRAMP4 (NCBI accession number AB525887) can selectively regulate the absorption of divalent metal cadmium (Cd) by rice roots and the transport of Cd from rice roots to aboveground parts. Inactivation of the OsNRAMP4 gene can significantly reduce cadmium accumulation in rice, but has almost no effect on the absorption and transport of manganese (Mn). Therefore, the NRAMP4 gene has the potential to be used as a genetic resource for crop variety improvement and breeding of new low-cadmium crop varieties. Attached Figure Description

[0028] Figure 1 The phylogenetic tree of the natural resistance-associated macrophage family (NRAMP) and its relative expression levels in the root region are shown. Where A: Phylogenetic tree of the natural resistance-associated macrophage family; B: Relative expression level of the natural resistance-associated macrophage family in the root region.

[0029] Figure 2 The predicted structures of OsNRAMP5 and OsNRAMP4 proteins and their binding sites with manganese ions are shown in Figure 3. A and B represent the predicted structures of OsNRAMP5 and OsNRAMP4 proteins by Alphafold3; C and D represent the predicted binding sites of OsNRAMP5 and OsNRAMP4 with manganese ions by Alphafold3.

[0030] Figure 3The mutation strategy and mutation information of the OsNRAMP4 mutant are shown. A: nramp4-1 and nramp4-2 are a Tos17 exon insertion mutant and a frameshift mutant with a 2bp exon deletion, respectively; B: RT-PCR identification of the nramp4-1 mutant genotype; C: Genotyping identification of the nramp4-2 mutant.

[0031] Figure 4 This chart displays the hydroponic phenotypes of wild-type japonica rice Nipponbare (NPB), indica rice Kasa, and mutant strains nramp4-1 and nramp4-2, as well as the cadmium and manganese content in the roots and aboveground parts of the plants. In the chart, A and D represent the hydroponic phenotypes of rice; B is a bar chart of cadmium content in the aboveground parts (shoot); C is a bar chart of manganese content in the aboveground parts; E is a bar chart of cadmium content in the roots; and F is a bar chart of manganese content in the roots. Detailed Implementation

[0032] In our ongoing research on the cadmium accumulation mechanism in rice, we unexpectedly discovered that the known tunable trivalent aluminum ion Al... 3+ The transmembrane aluminum ion transporter OsNRAMP4 (also labeled OsNRAT1, NCBI accession number AB525887) can also selectively regulate divalent cadmium ions (Cd). 2+ Transmembrane transport of divalent metal ions (such as Fe2+, Mn2+, Cd2+) has not yet been reported in the literature, and there is no literature reporting whether it can regulate the transport of divalent metal ions (such as Fe2+, Mn2+, Cd2+). 2 The transport of cadmium (C) and manganese (Mn) ions. We knocked out / inactivated the OsNRAMP4 gene in the genomes of wild-type japonica rice Nipponbare (NPB) and indica rice Kasa, and found that cadmium accumulation was significantly reduced in the mutants, but manganese (Mn) accumulation was almost not reduced. Based on these results, it can be inferred that the Nramp aluminum ion transporter gene can be used as a genetic resource to modify wild-type plants to reduce cadmium accumulation in more plant species and to cultivate new plant varieties with low cadmium accumulation.

[0033] The term "OsNRAMP4" refers to natural resistance-associated macrophage protein 4, a member of the natural resistance-associated macrophage protein (NRAMP or Nramp) family. The NRAMP family is a highly conserved family of transmembrane transport proteins, possessing typical transmembrane domains (usually containing 10-12 transmembrane helices). Several members transport divalent metal ions (such as Fe2+, Mn2+, and Cd) via proton coupling mechanisms. 2 (+, etc.) participate in the steady-state regulation of metal ions.

[0034] As used in this article, the term "wildtype" refers to native plants that have a normal phenotype and express normal Nramp aluminum ion transporter genes (such as the rice-derived OsNRAMP4 gene), such as Japonica rice Nipponbare (NPB) and Indica rice Kasa.

[0035] Correspondingly, the terms "low cadmium accumulation plants," "transgenic plants," and "genetically engineered plants" in this article have the same meaning, all referring to plants that have low cadmium accumulation after being genetically engineered from wild-type plants or plants with the original normal biological phenotype.

[0036] In some implementations, the term "(cadmium accumulation or Cd content) reduction" or "reduction" can mean a decrease of at least 10% compared to a reference level (such as normal plants), for example, a decrease of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or up to and including a decrease of 100%, or any decrease between 10% and 100%, or a decrease of at least about 2 times, or at least about 3 times, or at least about 4 times, or at least about 5 times, or at least about 10 times compared to a reference level.

[0037] In this document, for the sake of simplicity, the names of a protein, such as OsNRAMP4, and its encoding gene (DNA) are sometimes used interchangeably. Those skilled in the art should understand that they represent different substances in different descriptive contexts. Their meanings are readily understood by those skilled in the art based on the context. For example, when describing the function or class of the Nramp aluminum ion transporter, OsNRAMP4 refers to the protein; when described as a gene, it refers to the gene encoding that protein.

[0038] There are various techniques for inactivating, attenuating, and / or preventing the expression of Nramp aluminum ion transporters such as OsNRAMP4 in plants. These techniques can be used individually or in combination. For example, one inactivation method involves mutating the gene encoding the Nramp aluminum ion transporter, causing an alteration in the amino acid sequence of the polypeptide and / or termination of translation.

[0039] In the description of the technical solutions of this invention, the term "and / or" used in terms such as "A and / or B" or "A and / or B" is intended to include both A and B; A or B; A (alone); and B (alone). Similarly, the term "and / or" used in phrases such as "A, B, and / or C" is intended to cover each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); C (alone).

[0040] Based on the research of this invention, the OsNRAMP4 gene functional mutant can be used to improve the agronomic traits of rice grown under cadmium pollution conditions. The improved agronomic traits include: (a) reducing the cadmium content in the aboveground parts of rice; and (b) reducing the cadmium content in the roots of rice.

[0041] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the invention.

[0042] Example

[0043] The examples involve the addition amount, content and concentration of various substances, and unless otherwise specified, the percentage content refers to the mass percentage content.

[0044] In the embodiments, if the operating temperature is not specifically specified, it generally refers to room temperature (15-35°C).

[0045] Materials and general methods

[0046] The gene sequencing and primer synthesis in this embodiment were outsourced to Sangon Biotech (Shanghai) Co., Ltd.

[0047] PCR amplification experiments should be performed according to the reaction conditions provided by the plasmid or DNA template supplier or the kit instructions. Adjustments can be made through simple experiments if necessary.

[0048] Molecular biology methods, including gene editing technology, and methods for constructing transgenic plants are operated using techniques commonly used in the field.

[0049] plant materials

[0050] The nramp4-1 mutant used in this study was provided by the laboratory of Jianfeng Ma at Okayama University, Japan, and the variety is Nipponbare (NPB).

[0051] The nramp4-2 mutant was obtained in our laboratory using CRISPR / Cas9 technology, and the variety is Kasalath (Kasa for short).

[0052] The sgRNA primers constructed from the CRISPR / Cas9 plasmid that knocked out OsNRAMP4 are as follows:

[0053] 5'-ACTATTGACAGTGAAAAAGA TGG -3', and

[0054] 3'-CCATCTTTTCACTGTCAATAGT-5'.

[0055] Genetic transformation

[0056] 1. Callus Induction: Select mature and plump rice seeds, remove the husk; disinfect with 75% alcohol for 1-2 minutes, then discard the alcohol; rinse twice with sterile distilled water; soak in 0.15% mercuric chloride (containing 0.1% Tween 20) for 15-18 minutes, shaking several times during this period; discard the mercuric chloride, and rinse five times with sterile distilled water. Inoculate the sterilized seeds into the callus induction medium and culture at 32℃ under light for 5-10 days.

[0057] 2. Agrobacterium streak activation: Two days before infection, Agrobacterium was streaked on LB medium containing 50 mg / L kanamycin and incubated at 28°C.

[0058] 3. Agrobacterium suspension, infection, and co-culture: Before infection, activated Agrobacterium was scraped into suspension medium and cultured at 28°C with shaking at 180 rpm for 3–3.5 h. The bacterial concentration was then adjusted to OD600 = 0.1–0.2 using suspension medium. Callus tissue induced for 5–10 days was placed in the Agrobacterium suspension and infected for 1.5 min. The bacterial suspension was discarded, and the surface of the callus was blotted dry with sterile filter paper. The callus surface was covered with sterile filter paper and dried in a laminar flow hood for 30 min. After drying, the callus was transferred to co-culture medium covered with a layer of sterile filter paper, incubated overnight at 20°C in the dark, and then transferred to a 25°C incubator for further dark incubation for 2 days.

[0059] 4. Cleaning: After co-culturing, transfer the callus tissue to an empty sterile container using forceps. Wash the callus repeatedly with sterile distilled water 7-8 times, with the first 3 washes being quick and the subsequent 3-4 washes involving soaking for 3-5 minutes each. Finally, soak the callus in sterile distilled water containing 500 mg / L Cn (Carbenicillin) for 30 minutes. Discard the solution, blot the surface of the callus as dry as possible with sterile filter paper, cover the callus surface with another layer of sterile filter paper, and air dry in a laminar flow hood for 1 hour.

[0060] 5. Screening: Place the cleaned callus on the screening medium and incubate at 32°C under light for 14 days.

[0061] 6. Differentiation: After 14 days of screening, the resistant callus was transferred to differentiation medium and cultured at 28°C (photocycle of 14h light / 10h dark).

[0062] 7. Rooting: When the resistant callus forms a 3-4 cm tall regenerated seedling on the differentiation medium, transfer it to the rooting medium for culture until a complete plant is formed.

[0063] hydroponic experiment

[0064] Seeds from wild-type, nramp4-1, and nramp4-2 lines were soaked in water at 37°C for 2 days, then transferred to a net at 25°C containing a 0.5 mM CaCl2 solution. After 2 weeks, the plants were transferred to a 1 / 2 Kimura B nutrient solution containing 0.1 μM CdCl2. The pH of the nutrient solution was adjusted to 5.5, and the solution was changed every 3 days. The plants were grown in a climate chamber with 12 hours of light at 28°C and 12 hours of darkness at 22°C. After 2 weeks, different genotypes were photographed for comparison, and then the fresh weight of the aboveground parts and roots of the plants was measured.

[0065] Determination of metal content in plant tissues

[0066] Plant tissues, including roots, aerial parts, and seeds, were dried at 65°C for 7 days. A portion of the dried sample was weighed, digested with concentrated HNO3 / HClO4 (85:15, v / v), heated at 90°C for 1 h, then at 105°C for 3 h, and finally diluted to 10 mL with ultrapure water. The concentration of Cd in the solution was determined by inductively coupled plasma mass spectrometry (ICP-MS; PerkinElmerNexION300D).

[0067] Example 1: Analysis of NRAMP Family Members

[0068] We are involved in the reaction of divalent metal ions (such as Fe2+, Mn2+, Cd) 2 An analysis was conducted on NRAMP family members used in (+, etc.) transportation. See also Figure 1 The phylogenetic tree of the NRAMP family of natural resistance-associated macrophage proteins and their relative expression levels at the root.

[0069] Rice absorbs cadmium from the soil primarily through cadmium / manganese transport proteins in its roots. The OsNRAMP4 gene encodes natural resistance-associated macrophage protein 4 (…). Figure 1 (A) Natural resistance-associated macrophages are a highly conserved family of transmembrane transporters. Other members of this family, such as OsNRAMP5, OsNRAMP1, and OsNRAMP2, have been reported to participate in manganese and cadmium transport in rice, with OsNRAMP5 considered the most important transporter mediating manganese / cadmium uptake in rice roots (Sasaki et al., 2012). OsNRAMP4 mRNA shows high expression levels in rice roots, significantly higher than other members of its family. Figure 1 (B)

[0070] Example 2: Prediction of the protein structures of OsNRAMP5 and OsNRAMP4 and their manganese ion binding sites

[0071] We systematically predicted the Mn2+ binding capacity and coordination characteristics of rice natural resistance-related macrophage proteins OsNRAMP5 and OsNRAMP4 using the AlphaFold3 multimer model. (See also...) Figure 2 The results showed that both transporters exhibited high-confidence three-dimensional structure predictions (OsNRAMP5: ipTM = 0.93, pTM = 0.78; OsNRAMP4: ipTM = 0.92, pTM = 0.77), and their transmembrane core domains showed significant structural conservation, suggesting that they may have retained similar ion transport mechanisms during evolution. The prediction model indicated that the Mn of OsNRAMP5... 2+ The binding site is located in the extracellular loop region, with key coordinating residues Ala57 and Asp60 (residue confidence > 0.9). This region is known to be involved in OsNRAMP5-mediated Cd binding. 2 The Mn2+ uptake functions are highly compatible, and Asp60, as a polar amino acid, may participate in metal ion coordination through its carboxylate group. Compared to OsNRAMP5, OsNRAMP4 forms a more complex Mn2+ coordination network, involving four key residues: Gly64, Asp67, Ser69, and Asn70. Figure 2 ).

[0072] Example 3: Investigation of OsNRAMP4 gene inactivation mutants

[0073] To investigate the interaction between OsNRAMP4 and divalent manganese ions Mn 2+ To investigate whether there is a relationship between transport, we obtained OsNRAMP4 gene inactivation / knockout mutants through two methods.

[0074] The first mutant was created by inserting the tos-17 retrotransposon into the coding region of the OsNRAMP4 gene in the Nipponbare (NPB) genome, which caused translation errors in the protein, preventing it from encoding the correct protein. The resulting mutant line was named nramp4-1.

[0075] The second mutant was obtained by our laboratory using CRISPR / Cas9 technology to perform a frameshift mutation on the coding region of the OsNRAMP4 gene based on Kasalath (Kasa) indica rice, which caused premature termination of translation of the OsNRAMP4 protein. The resulting mutant line was named nramp4-2.

[0076] The mutant nramp4-1, provided by the laboratory of Jianfeng Ma at Okayama University, Japan, inserts the tos-17 retrotransposon into the seventh exon of the coding region (SEQ ID NO:1) of OsNRAMP4 (NCBI accession number AB525887), resulting in the OsNRAMP4 protein not being properly translated. Figure 3 A, B). We confirmed that nramp4-1 is a homozygous insertion mutant using primers flanking tos-17 (Xia et al., 2010). The primer sequences are as follows:

[0077] Forward primer: 5′-TGATGACATTCCTGAAGGTTGA-3′

[0078] Reverse primer: 5′-CGGAAACAAGATAGGGTCAAAC-3.

[0079] The mutant nramp4-2 was designed by our research group and involves a two-base AA deletion mutation at positions 113 and 114 of the first exon in the OsNRAMP4 gene coding region (SEQ ID NO:1), resulting in premature termination of translation of the OsNRAMP4 protein. Figure 3 (A, C). To this end, a knockout plasmid for CRISPR / Cas9 technology was constructed, transformed into Agrobacterium tumefaciens EHA105, and then transformed into wild-type rice callus using the Agrobacterium-callus transformation method, ultimately achieving knockout of the OsNRAMP4 gene. Sequencing confirmed the acquisition of the nramp4-2 mutant line. The construction process of the nramp4-2 mutant is briefly described below.

[0080] 1. Design knockout target sequences using the online website http: / / skl.scau.edu.cn / home / . Select the following sequences as target sequences:

[0081] 5'-ACTATTGACAGTGAAAAAGATGG-3', and

[0082] 3'-CCATCTTTTCACTGTCAATAGT-5'.

[0083] 2. The target sequence was introduced into the U6a promoter using the Golden Gate ligation method to construct a gRNA expression system;

[0084] 3. Perform PCR amplification of this fragment using the following primers:

[0085] B1':TTCAGAggtctcTctcgACTAGTGGAATCGGCAGCAAAGG, and

[0086] BL:AGCGTGggtctcGaccgACGCGTCCATCCACTCCAAGCTC.

[0087] After recovering the PCR fragments, the gRNA expression system was ligated into the Cas9 vector using Golden Gate ligation.

[0088] 4. After the vector was verified by sequencing, it was transformed into Agrobacterium EHA105 strain;

[0089] 5. The vector was transformed into rice callus tissue using Agrobacterium-mediated transformation to obtain CRISPR transgenic rice.

[0090] The obtained transgenic rice was identified as the nramp4-2 mutant line at the genomic and transcriptional levels.

[0091] Example 4: Investigation of agronomic traits of wild-type and mutant lines

[0092] To investigate whether the OsNRAMP4 mutation affects cadmium (Cd) uptake, wild-type, mutant nramp4-1, and nramp4-2 lines were subjected to hydroponic experiments, and cadmium was added to the nutrient solution to a concentration of 0.1 μM. Figure 4 (A and B). After two weeks of hydroponics in a cadmium-containing nutrient solution, samples were taken from the aboveground parts and roots, dried, and the cadmium content in the plant samples was determined. The results showed that significantly less cadmium accumulated in the aboveground parts of mutants nramp4-1 and nramp4-2. Figure 4 In the middle (C), the cadmium content of nramp4-1 and nramp4-2 in the roots was also significantly lower than that of the wild type (C). Figure 4 (D). Meanwhile, there was no significant difference in manganese content between the roots and aerial parts of the mutants nramp4-1 and nramp4-2. Figure 4 (E, F) The results show that OsNRAMP4 is involved in cadmium uptake in rice, and knocking out OsNRAMP4 significantly reduces cadmium content in rice roots and shoots. However, knocking out the OsNRAMP4 gene has almost no effect on manganese ion uptake and accumulation, suggesting that the OsNRAMP4 gene appears to have some specificity / selectivity in regulating the transmembrane transport of divalent metal ions.

[0093] Experimental data show that knocking out the OsNRAMP4 gene can significantly reduce the absorption and storage of cadmium in the roots and reduce the translocation of cadmium to the aboveground parts. Therefore, knocking out OsNRAMP4 has application value in low-cadmium rice breeding.

[0094] The above are merely preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be considered as limitations on the present invention, and the scope of protection of the present invention should be determined by the scope defined in the claims. For those skilled in the art, several improvements and modifications can be made without departing from the spirit and scope of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The use of Nramp aluminum ion transporter, a natural resistance-associated macrophage protein, to reduce cadmium accumulation in plants.

2. The use as described in claim 1, which is a method for reducing cadmium accumulation in plants by targeting Nramp aluminum ion transporter protein, characterized in that, Includes the following steps: It can downregulate, inactivate, weaken or knock out the Nramp aluminum ion transporter gene NRAMP4 in the chromosomes of wild-type plants.

3. The use as described in claim 2, characterized in that, The Nramp aluminum ion transporter is rice-derived Nramp aluminum ion transporter OsNRAMP4 (NCBI accession number AB525887), and the nucleotide sequence of its expression gene OsNRAMP4 coding region, namely CDS, is SEQ ID NO:

1.

4. The use as described in claim 2, characterized in that, Implemented in the following manner: (1) Knock out the Nramp aluminum ion transporter gene NRAMP4 in the chromosomes of wild-type plants; (2) Downregulate the expression level of the gene NRAMP4 in the chromosomes of wild-type plants; (3) Replace the NRAMP4 gene on the chromosome of wild-type plants with a mutant of the NRAMP4 gene that has lost or downregulated function; and / or (4) Block, inhibit or interfere with the expression of the gene NRAMP4 in the chromosomes of wild-type plants.

5. The use as described in claim 4, characterized in that, Method (2) is selected from the following group: (2-1) Mutations in the promoter region and / or coding region of the NRAMP4 gene lead to downregulation of the expression level of the NRAMP4 gene; (2-2) Mutations in the upstream regulators of the NRAMP4 gene lead to a downregulation of NRAMP4 expression; or (2-3) Introduce interacting proteins of Nramp aluminum ion transporter into wild-type plants to alter the function of the NRAMP4 gene.

6. The use as described in claim 5, characterized in that, The mutation in the coding region described in method (2-1) is a frameshift mutation, which leads to the inactivation or loss of function of the Nramp aluminum ion transporter; or it is the insertion of a retrotransposon in the coding region, which causes the protein to be translated incorrectly and cannot encode the correct protein.

7. The use as described in claim 6, characterized in that, When the plant is rice, the mutation in the coding region described in method (2-1) is a frameshift mutation, involving a two-base deletion mutation at positions 113 and 114 in the coding region of the OsNRAMP4 gene (NCBI accession number AB525887), i.e., the CDS nucleotide sequence SEQ ID NO:1, leading to premature termination of translation of the OsNRAMP4 protein; or The mutation in the coding region described in method (2-1) is the insertion of the tos-17 retrotransposon into the coding region of the gene OsNRAMP4 (NCBI accession number AB525887), i.e., the CDS nucleotide sequence SEQ ID NO:

1.

8. The use as described in claim 2, characterized in that, The steps are implemented through gene editing technology, antisense nucleic acids, and transcriptional regulation.

9. The use as described in claim 1, characterized in that, The plant is selected from the group consisting of: rice, wheat, corn, soybean, barley, oats, rye, sorghum, cotton, vegetables, tobacco, and cruciferous plants, with rice being the preferred plant.

10. The use as described in claim 1, characterized in that, Using the Nramp aluminum ion transporter gene as a target, we can cultivate plant varieties with low cadmium accumulation.

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