Method for improving the nickel accumulation capacity of plants and application in the remediation of nickel-contaminated soils

Abstract

The application discloses a method for improving the nickel enrichment capacity of plants and application in repairing nickel contaminated soil. O. chalcidica The two key FPN proteins and genes in the application are introduced into non-nickel hyperaccumulating plants, which significantly improves the absorption and root-to-shoot transport efficiency of plants to nickel, enhances the enrichment capacity of the shoots of plants to nickel, realizes the synergistic optimization of nickel transport and isolation functions, provides a new technical solution of high efficiency, low carbon and green environmental protection for the phytoremediation of nickel contaminated soil, and is suitable for the technical field of plant heavy metal remediation and resource recovery. In combination with high biomass plants or local dominant plants in areas with nickel pollution, more excellent nickel hyperaccumulating plant resources can be bred, regional green remediation of nickel contaminated soil is realized, and the application of a low-carbon and ecologically friendly nickel recovery strategy is promoted.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and more specifically, to a method for improving the nickel accumulation capacity of plants and its application in the remediation of nickel-contaminated soil. Background Technology

[0002] Nickel (Ni) is a crucial metallic element in the manufacture of industrial products such as stainless steel, alloys, and batteries. Mining activities can lead to excessive nickel levels, causing widespread pollution in agricultural and industrial land. Existing phytoremediation technologies for treating nickel-rich and mining-contaminated soils face challenges such as low nickel accumulation capacity of plants, limited detoxification efficiency, and restrictions on applicable species. Therefore, there is an urgent need to develop efficient and sustainable technologies for remediating nickel-contaminated soils.

[0003] Nickel is an essential micronutrient in the nitrogen cycle of plants, but when its concentration is too high, it can cause toxic stress and induce various molecular response mechanisms to regulate the transport, isolation, and detoxification of nickel within the plant. This applies to plants in the genus *Cephalotaxus* of the Brassicaceae family. Odontarrhena chalcidica ( O. chalcidica This plant is a typical nickel hyperaccumulator, with its aboveground tissues selectively accumulating up to 20,000 mg / kg of nickel, 20 times the hyperaccumulation standard (1,000 mg / kg). Furthermore, the nickel oxide (NiO) content in its biominerals exceeds 20%, far higher than the generally lower than 1.5% content in lateritic nickel ores. O. chalcidica It possesses strong metal selectivity, and during the enrichment of nickel, it does not co-enrich other metal elements such as zinc and cadmium, making it an ideal plant material for studying the specific enrichment mechanism of nickel. Currently, those skilled in the art... O. chalcidica Our understanding of this plant is extremely limited, and the exploration of the molecular mechanisms and key genes involved in nickel accumulation is still in its early stages. Furthermore, O. chalcidica Its poor ecological adaptability also limits its widespread application in the remediation of nickel-contaminated soil. Summary of the Invention

[0004] To address the aforementioned problems in existing technologies, this invention provides a method for enhancing the nickel accumulation capacity of plants and its application in the remediation of nickel-contaminated soil, aiming to explore nickel hyperaccumulating plants. O. chalcidica To investigate the functional differentiation and synergistic mechanism of FPN protein family members, construct transgenic plants with multi-gene synergistic expression, enhance the plant's ability to enrich nickel, and promote the absorption, translocation and accumulation of nickel, providing efficient, safe and scalable genetic engineering technology support for phytoremediation of heavy metal contaminated soils.

[0005] The first objective of this invention is to provide a method for improving the nickel enrichment capacity of plants.

[0006] The second object of the present application is to provide the application of the plant treated by the method in repairing nickel contaminated soil.

[0007] The third object of the present application is to provide the application of OcFPN1 protein and OcFPN2;1 protein.

[0008] The fourth object of the present application is to provide the application of OcFPN1 gene and OcFPN2;1 gene.

[0009] The fifth object of the present application is to provide the application of the biomaterials for promoting the expression of OcFPN1 protein and OcFPN2;1 protein.

[0010] The sixth object of the present application is to provide the application of the biomaterials for promoting the expression of OcFPN1 gene and OcFPN2;1 gene.

[0011] In order to achieve the above objects, the present application is realized by the following scheme:

[0012] In non-nickel-enriched plants, members of the FPN protein family are mainly responsible for isolating and detoxifying nickel in the underground part, and inhibiting the transport and accumulation of nickel in the aboveground part. The present application first discovers two members of the FPN protein family (OcFPN1 and OcFPN2;1) derived from nickel hyperaccumulating plants Odontarrhena chalcidica which have obviously different expression distribution and regulation function from the homologous FPN protein in Arabidopsis. OcFPN1 and OcFPN2;1 respectively play different roles in the transport, isolation and detoxification of Ni. OcFPN1 is mainly distributed in the underground part and has the ability to enhance the transmembrane transport of nickel; while OcFPN2;1 is mainly distributed in the aboveground part and has the function of cell nickel isolation and detoxification, and is responsible for isolating and detoxifying a large amount of nickel in the aboveground cells and accumulating a large amount of nickel.

[0013] At present, the research on FPN transport proteins in nickel hyperaccumulating plants mainly focuses on the function of a single gene, and lacks the research on the synergistic effect of multiple family members in the enrichment process. At the same time, there is no systematic functional verification method to determine the division mechanism of these proteins in the process of nickel transport and detoxification in vivo, resulting in the lack of effective molecular target and strategy support for the artificial construction of high nickel-enriched plants. Based on the characteristics and expression patterns of OcFPN1 gene and OcFPN2;1 gene, the present application further constructs single-gene heterologous expression and double-gene heterologous expression transgenic plants, detects the improvement effect of transgenic plants in nickel enrichment amount, transport efficiency and plant growth performance through metal treatment experiment, explores the molecular mechanism of the synergistic expression of multiple FPN genes, and determines that it can significantly improve the nickel transport efficiency and tolerance of plants under high Ni stress, and reveals that the twoO. chalcidica The FPN protein has a synergistic regulation effect in the mechanism of Ni hyperaccumulation, and can improve the nickel enrichment and repair ability of plants.

[0014] In conclusion, the application provides a systematic nickel enrichment molecular mechanism analysis and engineering strategy, which not only provides theoretical support for the efficient application of plant repair technology in nickel-contaminated soil, but also provides a resource basis for the development and promotion of related gene modules, and has important significance in the field of ecological environment protection and green repair.

[0015] Therefore, the application claims the following content:

[0016] A method for improving the nickel enrichment capacity of a plant, wherein a heterologous expression of OcFPN1 a gene and / or an OcFPN1 protein, and a heterologous expression of OcFPN2;1 a gene and / or an OcFPN2;1 protein is carried out in the plant; and the plant is a plant other than Odontarrhena chalcidica .

[0017] In some specific embodiments, the plant is Arabidopsis thaliana.

[0018] In some specific embodiments, the OcFPN1 nucleotide sequence of the gene is shown as SEQ ID NO. 21, and the OcFPN2;1 nucleotide sequence of the gene is shown as SEQ ID NO. 22.

[0019] In some specific embodiments, the amino acid sequence of the OcFPN1 protein is shown as SEQ ID NO. 33, and the amino acid sequence of the OcFPN2;1 protein is shown as SEQ ID NO. 34.

[0020] Application of the plant obtained by the method in repairing nickel-contaminated soil.

[0021] Application of the OcFPN1 protein and the OcFPN2;1 protein in any one of (1) to (8); (1) improving the nickel enrichment capacity of a plant; (2) preparing a product for improving the nickel enrichment capacity of a plant; (3) improving the tolerance of a plant to nickel stress; (4) preparing a product for improving the tolerance of a plant to nickel stress; (5) creating a nickel hyperaccumulating plant; (6) preparing a product for creating a nickel hyperaccumulating plant; (7) cultivating a plant for repairing nickel-contaminated soil; (8) preparing a product for cultivating a plant for repairing nickel-contaminated soil; and the plant is a plant other than Odontarrhena chalcidica .

[0022] OcFPN1 a gene and OcFPN2;1Use of the genes in any one of (1) to (8); (1) increasing the nickel accumulation ability of a plant; (2) preparing a product for increasing the nickel accumulation ability of a plant; (3) increasing the tolerance of a plant to nickel stress; (4) preparing a product for increasing the tolerance of a plant to nickel stress; (5) creating a hyperaccumulator of nickel; (6) preparing a product for creating a hyperaccumulator of nickel; (7) cultivating a plant for remediating nickel-contaminated soil; (8) preparing a product for cultivating a plant for remediating nickel-contaminated soil; the plant being other than Odontarrhena chalcidica Arabidopsis thaliana.

[0023] Use of the biological material for promoting the expression of the OcFPN1 protein and the OcFPN2;1 protein in any one of (1) to (8); (1) increasing the nickel accumulation ability of a plant; (2) preparing a product for increasing the nickel accumulation ability of a plant; (3) increasing the tolerance of a plant to nickel stress; (4) preparing a product for increasing the tolerance of a plant to nickel stress; (5) creating a hyperaccumulator of nickel; (6) preparing a product for creating a hyperaccumulator of nickel; (7) cultivating a plant for remediating nickel-contaminated soil; (8) preparing a product for cultivating a plant for remediating nickel-contaminated soil; the plant being other than Odontarrhena chalcidica Arabidopsis thaliana.

[0024] Use of the biological material for promoting the expression of the OcFPN1 protein and the OcFPN2;1 protein in any one of (1) to (8); (1) increasing the nickel accumulation ability of a plant; (2) preparing a product for increasing the nickel accumulation ability of a plant; (3) increasing the tolerance of a plant to nickel stress; (4) preparing a product for increasing the tolerance of a plant to nickel stress; (5) creating a hyperaccumulator of nickel; (6) preparing a product for creating a hyperaccumulator of nickel; (7) cultivating a plant for remediating nickel-contaminated soil; (8) preparing a product for cultivating a plant for remediating nickel-contaminated soil; the plant being other than OcFPN1 Arabidopsis thaliana. OcFPN2;1 Odontarrhena chalcidica In some embodiments, the plant is Arabidopsis thaliana.

[0025] Preferably, the increasing the nickel accumulation ability of a plant comprises at least one of increasing the ability of a plant to absorb nickel from the soil, increasing the ability of nickel to be translocated from the below-ground parts of a plant to the above-ground parts, or increasing the ability of nickel to accumulate in the above-ground parts of a plant.

[0026] Preferably, the biological material is any one of (1) to (4): (1) a nucleic acid molecule composition consisting of a nucleic acid molecule 1 encoding the OcFPN1 protein and a nucleic acid molecule 2 encoding the OcFPN2;1 protein; (2) an expression cassette containing the nucleic acid molecule composition of (1); (3) a recombinant expression vector containing the expression cassette of (2); (4) a microorganism containing the recombinant expression vector of (3).

[0027] Preferably, the biological material is any one of (1) to (4): (1) a nucleic acid molecule composition consisting of a nucleic acid molecule 1 encoding the OcFPN1 protein and a nucleic acid molecule 2 encoding the OcFPN2;1 protein; (2) an expression cassette containing the nucleic acid molecule composition of (1); (3) a recombinant expression vector containing the expression cassette of (2); (4) a microorganism containing the recombinant expression vector of (3). ​

[0028] More preferably, in (1), the nucleotide sequence of the nucleic acid molecule 1 is as shown in SEQ ID NO. 21 or as shown in the complete complementary sequence of the sequence as shown in SEQ ID NO. 21, and the nucleotide sequence of the nucleic acid molecule 2 is as shown in SEQ ID NO. 22 or as shown in the complete complementary sequence of the sequence as shown in SEQ ID NO. 22.

[0029] In addition, the present application also utilizes OcFPN2;1 the native promoter of the gene in O. chalcidica , realizes high expression of the gene in the aerial part of Arabidopsis thaliana, provides an expression regulation element for spatial differentiation of nickel transport and isolation function, and has important application value for constructing efficient and controllable engineering plants. More preferably, in (2), the promoter of the nucleic acid molecule 1 in the expression cassette is a CaMV 35S promoter, and the NCBI number of the promoter of the nucleic acid molecule 2 is PV246834.

[0030] The present application does not have special limitations on the source and type of the recombinant expression vector of the biological material in (3), including but not limited to yeast expression vectors, Agrobacterium expression vectors and other recombinant expression vectors capable of expressing the nucleic acid molecule composition, which can achieve the purpose of the present application. Preferably, the recombinant expression vector in (3) includes a yeast expression vector or an Agrobacterium expression vector. More preferably, the yeast expression vector takes pYES2 vector as a skeleton. More preferably, the Agrobacterium expression vector takes pCAMBIA1305 vector or pCAMBIA1301 vector as a skeleton.

[0031] The present application does not have special limitations on the source and type of the microorganism in (4) of the biological material, including but not limited to yeast, Escherichia coli, Agrobacterium and other conventional microorganisms that can carry the recombinant expression vector, which can achieve the purpose of the present application. Preferably, the microorganism in (4) includes yeast or Agrobacterium. More preferably, the yeast is a BY4741 yeast strain. More preferably, the Agrobacterium is Agrobacterium GV3101.

[0032] Compared with the prior art, the present application has the following beneficial effects:

[0033] The present application realizes the high expression of the gene in the aerial part of Arabidopsis thaliana by O. chalcidicaThe genes encoding two key FPN proteins in the application are introduced into non-nickel hyperaccumulating plants, significantly improving the absorption and root-to-shoot transport efficiency of plants to nickel, enhancing the ability of plants to enrich nickel in the shoots, and achieving the synergistic optimization of nickel transport and isolation function, providing a new technical solution for the phytoremediation of nickel-contaminated soil, which is efficient, low-carbon and environmentally friendly, and is suitable for the related technical fields of plant heavy metal remediation and resource recovery. Combined with high biomass plants or local dominant plants in areas with nickel pollution, more excellent nickel hyperaccumulating plant resources can be expected to be cultivated, regional green remediation of nickel-contaminated soil can be realized, and the application of low-carbon and ecologically friendly nickel recovery strategies can be promoted.

[0034] The specific advantages of the application are as follows:

[0035] (1) The system reveals the function and regulation mechanism of FPN protein family in nickel hyperaccumulating plants and provides molecular elements related to Ni phytoremediation: The application clearly shows that OcFPN1 It has the ability to enhance nickel transmembrane transport and nickel detoxification in plants, OcFPN2;1 It has a nickel isolation function, which fills the gap in the prior art in understanding the mechanism of FPN protein in nickel hyperaccumulating plants. OcFPN1 The nickel transport activity is significantly stronger than that of FPN in non-nickel hyperaccumulating plants, which promotes nickel absorption ;OcFPN2;1 The promoter sequence is specific for expression in the shoots and can be used for directional regulation of plant expression in the shoots.

[0036] (2) Realize the construction of efficient transport system by synergistic expression of multiple genes, and improve the nickel enrichment capacity of plants: Unlike the prior art which relies on single gene modification, the application realizes the combination of transport, isolation and tolerance functions in model plants by constructing a synergistic expression system of multiple FPN proteins, effectively improving the absorption, transport and tolerance of plants to nickel, and improving the overall plant remediation efficiency. The transport factor of non-nickel hyperaccumulating plants to nickel is increased from 0.3 to 2.78, reaching the standard of nickel hyperaccumulating plants (TF>1); the total amount of nickel extraction in the shoots is increased by 110%; the tolerance under high nickel stress is significantly enhanced.

[0037] (3) Provide high nickel enrichment gene module resources suitable for improvement and regionalization of different species: The genetic engineering operation scheme provided by the application can be widely applied to different plant backgrounds, especially suitable for introducing local plants with high biomass and rapid growth, overcoming the problem of poor ecological adaptability and limited distribution range of current nickel hyperaccumulating plants, providing a feasible strategy for nickel pollution control in South Asia, East Asia, Africa and other regions. The molecular mechanism and function verification system established by the application can be used as a basic platform for constructing engineering plants, which can not only be used for plant remediation in the field of environmental governance, but also has application potential in sustainable resource recovery scenarios such as plant mining, and promotes the practical application of green and low-carbon technology. BRIEF DESCRIPTION OF DRAWINGS

[0038] Figure 1 for O. chalcidica FPN Comparison of gene expression levels in plant tissues with homologous genes in Arabidopsis thaliana; A represents... OcFPN1 Genes in O. chalcidica The expression levels of the aboveground and underground parts and AtFPN1 Gene expression levels in the aboveground and underground parts of Arabidopsis thaliana; B represents... OcFPN2;1 Genes in O. chalcidica The expression levels of the aboveground and underground parts and AtFPN2 Gene expression levels in the aboveground and underground parts of Arabidopsis thaliana; nd indicates not detected; Ns indicates... p >0.05, * indicates p <0.05.

[0039] Figure 2 Empty vector - yeast AtFPN1 - Yeast and OcFPN1 - Yeast growth under conditions with no Ni or with 1 mM Ni.

[0040] Figure 3 Empty vector - yeast AtFPN2 - Yeast and OcFPN2;1 - Yeast growth under conditions with no Ni or with 1 mM Ni.

[0041] Figure 4 The results show the quantitative PCR expression identification of transgenic Arabidopsis thaliana plants; A represents WT Arabidopsis thaliana. atfpn2 Arabidopsis thaliana and 4 plants atfpn2 / 35S- FPN1 In Arabidopsis thaliana OcFPN1 Gene expression status; B represents WT Arabidopsis thaliana. atfpn2 Arabidopsis thaliana and 4 plants atfpn2 / pFPN2;1- FPN2;1 Arabidopsis thaliana OcFPN2;1 Gene expression status; C represents WT Arabidopsis thaliana, atfpn2 Arabidopsis thaliana and 4 plants atfpn2 / Co- FPN Arabidopsis thaliana OcFPN1 Gene expression status; D represents WT Arabidopsis thaliana, atfpn2 Arabidopsis thaliana and 4 plants atfpn2 / Co- FPN Arabidopsis thaliana OcFPN2;1 Gene expression status; Ns. indicates p >0.05; the same lowercase letter indicates no significant difference, and different lowercase letters indicate a significant difference.

[0042] Figure 5 For WT Arabidopsis thaliana, atfpn2 Arabidopsis thaliana andatfpn2 / 35S- FPN1 Figure 17. The accumulation and translocation of nickel in shoots and roots of Ex Arabidopsis in the nickel treatment plate experiment; A is the phenotype of each plant; B is the statistical result of the nickel concentration in shoots and roots of each plant under the condition of 150 μΜ Ni concentration; C is the statistical result of the total nickel enrichment in shoots and roots of each plant under the condition of 150 μΜ Ni concentration; D is the statistical result of the translocation coefficient of each plant under the condition of 150 μΜ Ni concentration; Ns. indicates no significant difference, different lowercase letters indicate significant difference. p >0.05; the same lowercase letter indicates no significant difference, and different lowercase letters indicate significant difference.

[0043] Figure 6 WT Arabidopsis, atfpn2 Arabidopsis and atfpn2 / Co- FPN2;1 Figure 17. The accumulation and translocation of nickel in shoots and roots of Ex Arabidopsis in the nickel treatment plate experiment; A is the phenotype of each plant; B is the statistical result of the nickel concentration in shoots and roots of each plant under the condition of 150 μΜ Ni concentration; C is the statistical result of the total nickel enrichment in shoots and roots of each plant under the condition of 150 μΜ Ni concentration; D is the statistical result of the translocation coefficient of each plant under the condition of 150 μΜ Ni concentration; Ns. indicates no significant difference, different lowercase letters indicate significant difference. p >0.05; the same lowercase letter indicates no significant difference, and different lowercase letters indicate significant difference.

[0044] Figure 7 WT Arabidopsis, atfpn2 Arabidopsis and atfpn2 / Co- FPN Figure 17. The accumulation and translocation of nickel in shoots and roots of Ex Arabidopsis in the nickel treatment plate experiment; A is the phenotype of each plant; B is the statistical result of the nickel concentration in shoots and roots of each plant under the condition of 150 μΜ Ni concentration; C is the statistical result of the total nickel enrichment in shoots and roots of each plant under the condition of 150 μΜ Ni concentration; D is the statistical result of the translocation coefficient of each plant under the condition of 150 μΜ Ni concentration; Ns. indicates no significant difference, different lowercase letters indicate significant difference. p >0.05; the same lowercase letter indicates no significant difference, and different lowercase letters indicate significant difference. DETAILED DESCRIPTION

[0045] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods; the materials and reagents used, unless otherwise specified, are commercially available. The following embodiments use… O. chalcidica The germplasm resources were donated by Professor Mao Lei (Distinguished Professor of the School of Environmental Science and Engineering, Sun Yat-sen University), and are available to the public from the applicant.

[0046] Example 1 FPN Gene expression analysis

[0047] 1. Experimental Methods

[0048] To investigate O. chalcidica middle FPN The tissue-specific expression characteristics of genes, this embodiment focuses on... OcFPN1 and OcFPN2;1 Expression levels in the aboveground parts and roots were analyzed by qRT-PCR and compared with homologous genes in Arabidopsis thaliana (…). AtFPN1 and AtFPN2 Compare them separately. O. chalcidica Total RNA was extracted from the aboveground and underground (root) tissues of Arabidopsis thaliana, and genomic DNA contamination was removed by DNase I treatment. cDNA was then synthesized via reverse transcription using oligo(dT) primers and random primers. Quantitative PCR (qPCR) was performed using the primers shown in Table 1, with the obtained cDNA as a template. The reaction was performed on an Applied Biosystems QuantStudio 3 real-time PCR instrument (Thermo Fisher, USA), using SYBR Green fluorescent dye to prepare the reaction system. Two technical replicates were performed for each sample. Two... -ΔΔCT The method calculates the relative expression level of each gene to achieve relative quantitative analysis.

[0049] Table 1 Quantitative PCR Primers

[0050]

[0051] 2. Experimental Results

[0052] like Figure 1 As shown in A, OcFPN1 exist O. chalcidica The expression level in the roots was significantly higher than that in the shoots, while its Arabidopsis homologs... AtFPN1 No significant difference in expression was observed between the above-ground parts and the roots. Figure 1 As shown in B, OcFPN2;1 The expression pattern of its homologous genes in Arabidopsis thalianaAtFPN2 obviously different, AtFPN2 mainly expressed in the roots of Arabidopsis thaliana, and hardly expressed in the aerial parts; while OcFPN2;1 In O. chalcidica The expression level in the aerial parts was significantly higher than that in the roots, 71 times higher.

[0053] The above results show that, OcFPN1 genes and OcFPN2;1 The gene has different expression distribution trends with its homologous gene in Arabidopsis thaliana, in which OcFPN2;1 The aerial part-specific expression of the gene may be closely related to its function in the process of nickel accumulation.

[0054] Example 2 FPN Construction of recombinant expression vector of the gene and functional identification of the yeast system

[0055] 1、 FPN Construction of recombinant expression vector of the gene

[0056] The total RNA of the tissues (mixed samples of roots and aerial parts) of the wild type Arabidopsis thaliana (denoted as WT Arabidopsis thaliana) and the mutant Arabidopsis thaliana (denoted as mutant Arabidopsis thaliana) was extracted respectively, and cDNA was obtained by reverse transcription. Using the obtained cDNA as a template, the four groups of cloning primers shown in Table 2 were used in turn, and the corresponding O. chalcidica cloning product of the gene (the nucleotide sequence is shown as SEQ ID NO. 19), AtFPN1 cloning product of the gene (the nucleotide sequence is shown as SEQ ID NO. 20), AtFPN2 cloning product of the gene (the nucleotide sequence is shown as SEQ ID NO. 21), and OcFPN1 cloning product of the gene (the nucleotide sequence is shown as SEQ ID NO. 22) were obtained by PCR amplification, and after purification, they were connected to a cloning vector and identified by sequencing to be correct sequences. OcFPN2;1

[0057] Table 2 Cloning primers of the gene

[0058]

[0059] Based on the homologous recombination method, using one-step cloning kit (ClonExpress Ultra One Step Cloning Kit V2-C116, manufacturer: Nanjing Novozyme), the four groups of homologous recombination primers shown in Table 3 were used in turn, and the AtFPN1 cloning product of the gene (SEQ ID NO. 19), AtFPN2 cloning product of the gene (SEQ ID NO. 20), OcFPN1 cloning product of the gene (SEQ ID NO. 21), and OcFPN2;1 ​The cloning product of the gene (such as SEQ ID NO. 22) is connected between the Kpnl site and the BamHI site of the pYes2 yeast expression vector, and the yeast expression vector identified as expected by sequencing verification is obtained, that is, the corresponding recombinant expression vector of each gene. Among them, the expression AtFPN1 protein (the amino acid sequence is shown as SEQ ID NO. 31) is recorded as pYes2- AtFPN1 vector, and the expression AtFPN2 protein (the amino acid sequence is shown as SEQ ID NO. 32) is recorded as pYes2- AtFPN2 vector, and the expression OcFPN1 protein (the amino acid sequence is shown as SEQ ID NO. 33) is recorded as pYes2- OcFPN1 vector, and the expression OcFPN2;1 protein (the amino acid sequence is shown as SEQ ID NO. 34) is recorded as pYes2- OcFPN2;1 vector.

[0060] Table 3 Homologous recombination primers

[0061]

[0062] 2, Construction of yeast system

[0063] Based on the lithium chloride-PEG method, the empty vector (i.e. pYES2 vector), pYes2- 1 vector, pYes2- 2 vector, pYes2- 3 vector, pYes2- 4 vector and pYes2- 5 vector are respectively transformed into the yeast strain BY4741 (genotype: his3A leu2A met15A ura3A) by using the classic yeast transformation kit (manufacturer: Beijing Coolabotech Electronics Co., Ltd., product number: SK2400-200T), and the yeast strains expressing the functional AtFPN1 gene are screened and reserved, and the yeast strains expressing the AtFPN2 gene are screened and reserved, and the yeast strains expressing the OcFPN1 gene are screened and reserved, and the yeast strains expressing the OcFPN2;1 gene are screened and reserved, and the yeast strains expressing the ura3 gene are screened and reserved, and the yeast strains expressing the ura3 gene are screened and reserved, and the yeast strains expressing the AtFPN1 gene are screened and reserved, and the yeast strains expressing the AtFPN1 gene are screened and reserved, and the yeast strains expressing the AtFPN2 gene are screened and reserved, and the yeast strains expressing the AtFPN2-Yeast, transfer to pYes2- OcFPN1 The recombinant yeast vector is denoted as OcFPN1 -Yeast, transfer to pYes2- OcFPN2;1 The recombinant yeast vector is denoted as OcFPN2;1 -yeast.

[0064] 3. Functional identification

[0065] To assess the sensitivity of various recombinant yeasts to nickel, a drop assay was used for growth experiments. OD... 600 The culture solutions of each recombinant yeast at a concentration of 1.0 were prepared according to a series gradient (i.e., 1, 10). -1 10 -2 and 10 -3 After dilution, the recombinant yeasts were inoculated into SD-U agar medium containing 20 mM MES buffer (pH 5.5) (denoted as 0 mM nickel medium) and SD-U agar medium containing 1 mM NiCl2 and 20 mM MES buffer (pH 5.5) (denoted as 1 mM nickel medium), respectively, and cultured at 30°C. The colony growth status of each recombinant yeast was observed.

[0066] After 24 hours of cultivation, the empty vector-yeast culture medium grown in 0mM nickel medium... AtFPN1 -yeast, AtFPN2 -yeast, OcFPN1 - Yeast and OcFPN2;1 - Yeast and empty vector grown in 1 mM nickel medium - Yeast, AtFPN2 - Yeast and OcFPN2;1 - The yeast has reached a stable growth state; at this point, take a photograph for documentation. Empty vector-yeast is growing in 1mM nickel medium. AtFPN1 - Yeast and OcFPN1 -After the yeast continued to grow for 48 hours, it showed a stable growth state, at which point a photo was taken to record the growth.

[0067] like Figure 2 and Figure 3 As shown, under conditions without nickel (i.e., 0 mM Ni), the growth of the five recombinant yeast strains was similar, indicating that FPN protein expression itself does not affect the basal growth of yeast; however, under conditions with NiCl2 (i.e., 1 mM Ni), AtFPN1 The yeast showed only mild growth inhibition, while OcFPN1 Yeast, on the other hand, showed significantly stronger growth inhibition, indicating that... AtFPN1 Reduce yeast's sensitivity to nickel. OcFPN1 On the one hand, it enhances the yeast's sensitivity to nickel. OcFPN2;1 The yeast exhibited strong tolerance to Ni, and its growth was superior to that of the empty vector yeast. AtFPN2 The similarity in phenotypes of yeasts indicates that...AtFPN2 and OcFPN2;1 Both reduce the sensitivity of yeast to nickel.

[0068] The above results show that, OcFPN1 may promote the uptake of nickel by yeast and thus reduce the nickel tolerance of yeast, while OcFPN2;1 may play a role in the detoxification or isolation of nickel, thereby improving the nickel tolerance of yeast.

[0069] Example 3 Construction and functional identification of transgenic Arabidopsis plants

[0070] 1. Construction of transgenic Arabidopsis plants

[0071] (1) Construction of recombinant expression vector

[0072] OcFPN1 The recombinant expression vector of AtNRT2.1 was constructed as follows: The full-length coding sequence of AtNRT2.1 was ligated between the Kpnl site and the BamHI site of the plant expression vector pCAMBIA1305 using a one-step cloning kit and primers 35S-F (5'-gaacgataggagctcggtaccATGGAGAATGAGACAGAAGCAAAA-3' (SEQ ID NO. 35)) and 35S-R (5'-caggtcgactctagaggatccTTACATGTTTCCACGAGAAGAAAGC-3' (SEQ ID NO. 36)), with the CaMV 35S constitutive promoter driving the expression of the AtNRT2.1 gene. The vector was sequenced and verified to be consistent with the expected vector, and was recorded as 35S-AtNRT2.1. OcFPN1 OcFPN1 OcFPN1 FPN1 OcFPN2;1

[0073] OcFPN2;1 ​​​​​Recombinant expression vector of promoter: linearized pCAMBIA1301 was obtained by double digestion of plant expression vector pCAMBIA1301 with EcoRI and SacI, removing the original CaMV 35S constitutive promoter; homologous arms were added at both ends of the OcFPN2;1 promoter (NCBI: PV246834, denoted as pFPN2;1) using a one-step cloning kit and primers pFPN2;1-F (5'-tatgaccatgattacgaattcACCTATGCCAAAGAAGAACCAAA-3' (SEQ ID NO. 37)) and pFPN2;1-R (5'-ggatccccgggtaccgagctcGATTAAATCTGAACACAAACCCAAAA-3' (SEQ ID NO. 38)), and it was connected to the linearized pCAMBIA1301, and the vector that was identified as expected after sequencing verification was retained, denoted as p1301-pFPN2;1 vector.

[0074] OcFPN2;1 Recombinant expression vector of OcFPN2;1: linearized p1301-pFPN2;1 was obtained by double digestion of the p1301-pFPN2;1 vector with SacI and KSalI; the full-length coding sequence of OcFPN2;1 was connected to the downstream of pFPN2;1 in the linearized p1301-pFPN2;1 using a one-step cloning kit and primers OcFPN2;1 -F (5'-tgttcagatttaatcgagctcATGGACGAGGAAGGAGGAACTAG-3' (SEQ ID NO. 39)) and OcFPN2;1 -R (5'-gcatggaagatcttcgtcgacTTAGACACTACCTTGAGGTGTGTATCC-3' (SEQ ID NO. 40)), to drive the expression of FPN2;1 gene under the control of pFPN2;1, and the vector that was identified as expected after sequencing verification was retained, denoted as pFPN2;1-OcFPN2;1 vector. OcFPN1 Recombinant expression vector for combined expression: a one-step cloning kit and primers

[0075] OcFPN2;1 and Co- Recombinant expression vector for combined expression: a one-step cloning kit and primers FPN Co- -F1 (5'-tgttcagatttaatcgagctcATGGACGAGGAAGGAGGAACTAG-3' (SEQ ID NO. 39)), FPN Co-FPN-R1 (5'-tggagtagacTTGATGCATGTTGTCAATCAATTG-3' (SEQ ID NO.41)), Co-FPN -F2 (5'-catgcatcaaGTCTACTCCAAAAATATCAAAGATACAGTC-3' (SEQ ID NO. 42)) and OcFPN2;1 -R1(5'-aatgtttgaacgatcctgcagTTACATGTTTCCACGAGAAGAAAGC-3' (SEQ ID NO.43)) sequentially recombines pFPN2;1 through multi-fragment homologous recombination. OcFPN1 The full-length coding sequence, the CaMV35S constitutive promoter, FPN The full-length coding sequence is ligated between the SacI and PstI sites of the p1301-pFPN2;1 vector, and the vector that has been verified and confirmed to meet the expectations through sequencing is obtained, denoted as Co- FPN1 Carrier.

[0076] (2) Construction of Agrobacterium

[0077] Using the freeze-thaw method, 35S- Agrobacterium tumefaciens Transformation of vector into Agrobacterium tumefaciens ( OcFPN1 GV3101) obtained single expression FPN1 Agrobacterium, designated 35S- FPN2;1 Agrobacterium; pFPN2;1- OcFPN2;1 Transformation of the vector into GV3101 yielded single expression. FPN2;1 Agrobacterium, designated pFPN2;1- FPN Agrobacterium; Co- OcFPN1 The vector was converted to GV3101 to obtain OcFPN2;1 and FPN Co-expressed Agrobacterium is denoted as Co- AtFPN2 Agrobacterium.

[0078] (3) Infection and screening

[0079] The three Agrobacterium strains mentioned above were respectively infecting nickel-stress-sensitive... atfpn2 The knockout Arabidopsis mutant (Salk_074442, denoted as...) FPN1 Arabidopsis thaliana), the specific method is as follows: 35S- FPN2;1 Agrobacterium bacterial suspension, pFPN2;1- FPN Agrobacterium bacterial suspension and Co- FPN1 Agrobacterium bacterial suspensions were all based on initial OD values. 600 Add 0.8 μg of the bacterial culture solution to LB medium containing kanamycin and rifampin, respectively, and incubate at 28°C with shaking for 12 h. Centrifuge the bacterial culture and then adjust the OD value of the bacterial culture.600 0.8, resuspended in infiltration solution (ultrapure water containing 5% w / v sucrose and 0.02% Silwet-77) at a volume ratio of 2.5:1, to obtain 35S- Agro-infiltrated Arabidopsis plants containing 35S- Ex1, 35S- Ex2, 35S- Ex3 and 35S- Ex4, respectively. FPN2;1 Agrobacterium suspension (labeled as Infiltration solution 1), containing pFPN2;1- Agrobacterium suspension (labeled as Infiltration solution 2) and Co- Agrobacterium suspension (labeled as Infiltration solution 3). Healthy Arabidopsis thaliana plants were grown to the stage of bolting and flowering, and the siliques and pollinated flowers were removed before transformation. The inflorescence infection method was used to infect the Arabidopsis thaliana plants with Infiltration solution 1, and the plants were slightly shaken for 45 s; the infection was repeated three times with one-week intervals; the plants were continuously cultivated to maturity, and the seeds were collected, which were labeled as Seed 1. FPN1 Agrobacterium suspension (labeled as Infiltration solution 2) and Co- atfpn2 Agrobacterium suspension (labeled as Infiltration solution 3). Healthy Arabidopsis thaliana plants were grown to the stage of bolting and flowering, and the siliques and pollinated flowers were removed before transformation. The inflorescence infection method was used to infect the Arabidopsis thaliana plants with Infiltration solution 1, and the plants were slightly shaken for 45 s; the infection was repeated three times with one-week intervals; the plants were continuously cultivated to maturity, and the seeds were collected, which were labeled as Seed 1. atfpn2 Arabidopsis thaliana, which were grown to the stage of bolting and flowering, and the siliques and pollinated flowers were removed before transformation. The inflorescence infection method was used to infect the Arabidopsis thaliana plants with Infiltration solution 1, and the plants were slightly shaken for 45 s; the infection was repeated three times with one-week intervals; the plants were continuously cultivated to maturity, and the seeds were collected, which were labeled as Seed 1. FPN1 Arabidopsis thaliana, which were grown to the stage of bolting and flowering, and the siliques and pollinated flowers were removed before transformation. The inflorescence infection method was used to infect the Arabidopsis thaliana plants with Infiltration solution 1, and the plants were slightly shaken for 45 s; the infection was repeated three times with one-week intervals; the plants were continuously cultivated to maturity, and the seeds were collected, which were labeled as Seed 1. atfpn2 Agrobacterium-infiltrated Arabidopsis thaliana seeds, labeled as Seed 1. In the same way, pFPN2;1- Agrobacterium-infiltrated Arabidopsis thaliana seeds, labeled as Seed 2, and Co- Agrobacterium-infiltrated Arabidopsis thaliana seeds, labeled as Seed 3, were prepared. FPN2;1 Agrobacterium-infiltrated Arabidopsis thaliana seeds, labeled as Seed 1. In the same way, pFPN2;1- Agrobacterium-infiltrated Arabidopsis thaliana seeds, labeled as Seed 2, and Co- Agrobacterium-infiltrated Arabidopsis thaliana seeds, labeled as Seed 3, were prepared. atfpn2 Agrobacterium-infiltrated Arabidopsis thaliana seeds, labeled as Seed 1. In the same way, pFPN2;1- Agrobacterium-infiltrated Arabidopsis thaliana seeds, labeled as Seed 2, and Co- Agrobacterium-infiltrated Arabidopsis thaliana seeds, labeled as Seed 3, were prepared. FPN1 Agrobacterium-infiltrated Arabidopsis thaliana seeds, labeled as Seed 1. In the same way, pFPN2;1- Agrobacterium-infiltrated Arabidopsis thaliana seeds, labeled as Seed 2, and Co- Agrobacterium-infiltrated Arabidopsis thaliana seeds, labeled as Seed 3, were prepared. atfpn2 Agrobacterium-infiltrated Arabidopsis thaliana seeds, labeled as Seed 1. In the same way, pFPN2;1- Agrobacterium-infiltrated Arabidopsis thaliana seeds, labeled as Seed 2, and Co- Agrobacterium-infiltrated Arabidopsis thaliana seeds, labeled as Seed 3, were prepared. atfpn2 / Agrobacterium-infiltrated Arabidopsis thaliana seeds, labeled as Seed 1. In the same way, pFPN2;1- Agrobacterium-infiltrated Arabidopsis thaliana seeds, labeled as Seed 2, and Co- Agrobacterium-infiltrated Arabidopsis thaliana seeds, labeled as Seed 3, were prepared.

[0080] (4) Seed screening

[0081] Seeds 1-3 were screened by using 1 / 2MS medium containing 30 mg / L hygromycin, and the seeds with the ability to grow in the presence of hygromycin were retained and transplanted into nutrient soil for further cultivation. The corresponding plants were labeled as 35S- Ex Arabidopsis, pFPN2;1- Ex Arabidopsis and Co- Ex Arabidopsis, respectively. FPN1 35S- atfpn2 / Ex Arabidopsis, -FPN2;1 pFPN2;1 atfpn2 / Ex Arabidopsis and -FPN Co atfpn2 Ex Arabidopsis.

[0082] (5) Expression identification

[0083] For WT Arabidopsis, atfpn2 / Arabidopsis, 4 strains of 35S- FPN1 Ex Arabidopsis (labeled as 35S- Ex1, 35S- Ex2, 35S- Ex3 and 35S- Ex4, respectively), atfpn2 / 35S- FPN1 Ex1, atfpn2 / 35S- FPN1 Ex2, atfpn2 / 35S- FPN2;1 Ex3 and 35S- Ex4), atfpn2 / pFPN2;1- FPN2;1 Ex Arabidopsis (labeled as pFPN2;1- Ex1, pFPN2;1- Ex2, pFPN2;1- Ex3 and pFPN2;1- Ex4, respectively), atfpn2 / Ex1~ FPN2;1 pFPN2;1- atfpn2 / Ex4) and 4 plants FPN Co- atfpn2 / Arabidopsis thaliana (denoted as) FPN Co- atfpn2 / Ex1~ FPN Co- Figure 4 Ex4), total RNA was extracted from the aboveground and underground (i.e. root) tissues, respectively, and cDNA was obtained by reverse transcription. Using cDNA as a template, the gene expression of each transgenic Arabidopsis was identified using the primers in Table 4, referring to the quantitative PCR method in Example 1.

[0084] Table 4 Quantitative PCR Primers

[0085]

[0086] like atfpn2 / As shown in A, among 4 plants FPN1 35S- OcFPN1 In Arabidopsis thaliana, O. chalcidica The gene is expressed in both the above-ground and below-ground parts, but mainly in the below-ground parts, and is related to OcFPN1 middle Figure 4 The gene expression patterns are consistent. For example... atfpn2 / As shown in B, among 4 plants FPN2;1 pFPN2;1- OcFPN2;1 In Arabidopsis thaliana, atfpn2 / The gene is expressed in both the above-ground and underground parts, and except for FPN2;1 pFPN2;1- O. chalcidica Except for Ex1, which showed relatively high expression levels in the underground part (potentially indicating a significant nickel-segmenting effect in the underground part), the other three strains all showed high expression levels in the aboveground part. OcFPN2;1 middle Figure 4 The gene expression patterns are consistent. For example... atfpn2 / Co-FPN As shown in C and D, in 4 plants OcFPN1 In Arabidopsis thaliana, OcFPN2;1 Genes and OcFPN1 The gene is expressed in both the above-ground and underground parts, among which OcFPN2;1 The gene is still mainly expressed in the underground part. OcFPN2;1 The gene is also expressed at a higher level in the underground part, which is related to atfpn2 The patterns of heterologous expression of genes differ significantly.

[0087] 2. Identification of nickel accumulation capacity in transgenic Arabidopsis plants

[0088] (1) Nickel-treated plate experiment

[0089] To study the effect of FPN gene expression on the ability of Arabidopsis to enrich nickel, healthy and uniform T1 generation WT Arabidopsis, atfpn2 / Arabidopsis and FPN1 35S- atfpn2 Ex Arabidopsis were placed in 1 / 2 MS medium containing 5 mM MES (pH 5.5) buffer (referred to as regular medium) and 1 / 2 MS medium containing 150 μΜ NiCl2 and 20 mM MES (pH 5.5) buffer (referred to as regular nickel medium), respectively, with 4 parallel replicates of each Arabidopsis plant on each medium. Then, they were placed in an artificial climate chamber for 1 week, and the greenhouse culture conditions were as follows: 16 hours of light, 8 hours of darkness; day temperature of 25 °C, night temperature of 20 °C; relative humidity of 60%, and light intensity of 150 μmol / m 2 / s (PAR). The growth state of each plant in each medium was observed regularly during the culture.

[0090] To further study the nickel tolerance and nickel accumulation characteristics of the transgenic plants under higher nickel stress, healthy and uniform T1 generation WT Arabidopsis, atfpn2 / Arabidopsis and FPN2;1 pFPN2;1- atfpn2 Ex Arabidopsis were placed in the control medium, regular nickel medium, and 1 / 2 MS medium containing 250 μΜ NiCl2 and 20 mM MES (pH 5.5) buffer (referred to as high nickel medium), and were cultured according to the above method.

[0091] To further study the nickel tolerance and nickel accumulation characteristics of the transgenic plants under ultra-high nickel stress, healthy and uniform T1 generation WT Arabidopsis, atfpn2 / Arabidopsis and FPN Co- OcFPN1 Ex Arabidopsis were placed in the control medium, high nickel medium, and 1 / 2 MS medium containing 250 μΜ NiCl2 and 20 mM MES (pH 5.5) buffer (referred to as ultra-high nickel medium), and were cultured according to the above method.

[0092] (2) Determination of the biomass of the plants and the nickel concentration and transport efficiency

[0093] After the culture, the roots of each Arabidopsis plant were cut off at the root-stem junction with scissors, and the aerial part and root (i.e., underground part) of each plant were collected, respectively. The roots were placed in 50 mM Na2-EDTA solution under ice bath conditions to remove metal ions adsorbed on the root surface, and then were washed with pre-cooled ultrapure water. After the root system was cleaned, it was laid on paper and the surface water was absorbed by light pressing. The fresh weight of the aerial part and root system was weighed using an analytical balance, and the data were recorded, which was the biomass of each Arabidopsis plant.

[0094] The aboveground and underground parts of Arabidopsis thaliana plants grown in different culture media were weighed separately into digestion tubes. The plant tissues were digested in a graphite furnace digester at 105 °C using a USEPA 3050B system of concentrated nitric acid-hydrogen peroxide (4 mL 1:1 (w / v) HNO3 and 1 mL 30% (w / v) H2O2). The nickel concentration (μg kg) was then determined using inductively coupled plasma mass spectrometry (ICP-MS). -1 (FW). The total nickel accumulation of each Arabidopsis plant was obtained by multiplying the nickel concentration by the biomass. The differences in nickel accumulation between the aboveground and underground parts of each Arabidopsis plant were compared. The translocation factor (TF) was used to represent the efficiency of nickel translocation from the roots to the aboveground parts, TF = nickel concentration in the aboveground parts ÷ nickel concentration in the underground parts.

[0095] (3) Figure 5 Effects of heterologous gene expression on plants

[0096] like atfpn2 As shown in A, WT Arabidopsis thaliana and Figure 5 Root growth in Arabidopsis thaliana was significantly inhibited, while root growth in transgenic lines was not significantly inhibited, indicating that expression of OctFPN1 in Arabidopsis thaliana alleviates nickel toxicity. atfpn2 / As shown in B and C, all -FPN1 35S OcFPN1 The Ni concentration in the roots of *Arabidopsis thaliana* var. *extraterrestrialum* decreased significantly, with a reduction of 70.3% to 88.0%, indicating that... atfpn2 / It can effectively reduce Ni accumulation in the roots; and all -FPN1 35S OcFPN1 The Ni concentration in the aboveground parts of *Arabidopsis thaliana* ex was basically the same as that in the aboveground parts of *Arabidopsis thaliana* WT, indicating that... Figure 5 This ensures that the total Ni enrichment in the aboveground parts remains relatively stable. Regarding Ni translocation efficiency, such as... atfpn2 / As shown in D, all -FPN1 35S OcFPN1 The translocation coefficient of Arabidopsis thaliana was significantly increased, reaching 0.957–1.73, which is 5.0–7.1 times higher than that of WT Arabidopsis thaliana. These results indicate that... OcFPN2;1 The expression can alleviate nickel toxicity in Arabidopsis thaliana in the absence of root-isolated nickel and significantly enhance the root-to-shoot translocation efficiency of nickel, but is insufficient to increase the total enrichment of Ni in the shoots.

[0097] (4) Figure 6 Effects of heterologous gene expression on plants

[0098] like atfpn2 / As shown in A, at a concentration of 150 μM, except for-FPN2;1 pFPN2;1 atfpn2 / Ex1, the other 3 lines -FPN2;1 pFPN2;1 atfpn2 / Ex Arabidopsis growth was significantly inhibited; while under 250 μΜ, all -FPN2;1 pFPN2;1 Figure 6 Ex Arabidopsis growth was significantly inhibited. As shown in B and C of atfpn2 / , under 150 μΜ and 250 μΜ Ni, all -FPN2;1 pFPN2;1 atfpn2 / Ex Arabidopsis root Ni concentration was significantly lower than WT; while under 150 μΜ Ni, all -FPN2;1 pFPN2;1 atfpn2 / Ex Arabidopsis shoot Ni concentration showed no significant change; while under 250 μΜ Ni, all FPN2;1 pFPN2;1 - Figure 6 Ex Arabidopsis shoot Ni concentration was significantly increased by 34.1% - 41.1%, thus increasing total shoot Ni accumulation by 51.1% - 97.5%. In terms of Ni transport efficiency, as shown in D of atfpn2 / , under 150 μΜ Ni, all -FPN2;1 pFPN2;1 atfpn2 / Ex Arabidopsis transport factor was significantly increased; under 250 μΜ Ni, all -FPN2;1 pFPN2;1 atfpn2 / Ex Arabidopsis transport factor was further significantly increased to 0.630 - 0.678, but still lower than 1, failing to reach the standard of nickel hyperaccumulators (TF > 1), indicating ​ pFPN2;1 -FPN2;1 Ex Arabidopsis Ni accumulation was still mainly in the roots. The above results show that, OcFPN2;1 aboveground expression of

[0099] (5) OcFPN2;1 genes and OcFPN2;1 joint heterologous expression of genes on plants

[0100] As shown in A of Figure 7 , under 250 μΜ and 350 μΜ Ni, WT and atfpn2 root growth was significantly inhibited, while transgenic lines were not significantly inhibited, indicating that OcFPN1 andOcFPN2;1 Co-expression in Arabidopsis significantly alleviated nickel toxicity. For example... Figure 7 As shown in B, under Ni concentrations of 250 μM and 350 μM, all [various species] compared to WT Arabidopsis thaliana [were observed]. atfpn2 / Co -FPN The Ni concentration in the roots of *Arabidopsis thaliana* var. *ex* decreased significantly, ranging from 71.3% to 86.4%; under Ni concentration conditions of 250 μM, compared with *Arabidopsis thaliana* var. *w / w / *, all... atfpn2 / Co -FPN The Ni concentration in the aboveground parts of *Arabidopsis thaliana* decreased by 27.9%–34.6%, while under the condition of Ni concentration of 350 μM, all atfpn2 / Co -FPN The aboveground Ni concentration of *Arabidopsis thaliana* (Ex) was similar to that of *Arabidopsis thaliana* (WT). For example... Figure 7 As shown in C, although under the condition of Ni concentration of 250 μM, all atfpn2 / Co -FPN The total Ni accumulation in the aboveground parts of *Arabidopsis thaliana* ex was not significantly different from that in *Arabidopsis thaliana* WT, but it was significantly higher at a Ni concentration of 350 μM, with an increase of 48.3%–110%. Regarding Ni translocation efficiency, such as... Figure 7 As shown in D, under the condition of Ni concentration of 250 μM, compared with WT Arabidopsis, all atfpn2 / Co -FPN The translocation coefficient of *Arabidopsis thaliana* reached 1.28–1.55, an increase of 3.95–4.78 times, meeting the criteria for nickel hyperaccumulators (TF>1); under Ni concentration of 350 μM, all atfpn2 / Co -FPN The translocation coefficient of *Arabidopsis thaliana* exogene was further increased to 1.64–2.78, meeting the criteria for nickel hyperaccumulators (TF>1). These results indicate that... OcFPN1 and OcFPN2;1 The synergistic expression of the compound significantly enhances the nickel accumulation capacity of Arabidopsis thaliana, strengthens the efficiency of Ni translocation from roots to aboveground parts, especially under high concentration Ni stress, it increases the accumulation of nickel in the aboveground parts of the plant, and also significantly enhances the plant's nickel tolerance and alleviates the growth inhibition of the plant by nickel toxicity.

[0101] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description and ideas, and it is neither necessary nor possible to exhaustively describe all implementation methods here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A method for improving the ability of a plant to accumulate nickel, characterized in that, heterologously expressing in said plant OcFPN1 a gene, and heterologously expressing OcFPN2;1 a gene; said plant is Arabidopsis thaliana; The OcFPN1 The nucleotide sequence of the gene is shown in SEQ ID NO.

21. OcFPN2;1 The nucleotide sequence of the gene is shown in SEQ ID NO.

22.

2. The use of the plant treated by the method of claim 1 in remediating nickel-contaminated soil.

3. The use of the biological material for promoting the expression of OcFPN1 protein and OcFPN2;1 protein in any one of (1) to (8); (1) improving the nickel accumulation capacity of a plant; (2) preparing a product for improving the nickel accumulation capacity of a plant; (3) improving the tolerance of a plant to nickel stress; (4) preparing a product for improving the tolerance of a plant to nickel stress; (5) creating a nickel hyperaccumulator plant; (6) preparing a product for creating a nickel hyperaccumulator plant; (7) cultivating a plant for remediating nickel-contaminated soil; (8) preparing a product for cultivating a plant for remediating nickel-contaminated soil; the plant is Arabidopsis thaliana; the biological material is any one of (1) to (4): (1) a nucleic acid molecule composition consisting of a nucleic acid molecule 1 encoding the OcFPN1 protein and a nucleic acid molecule 2 encoding the OcFPN2;1 protein; (2) an expression cassette containing the nucleic acid molecule composition in (1); (3) a recombinant expression vector containing the expression cassette in (2); (4) a microorganism containing the recombinant expression vector in (3). The OcFPN1 The nucleotide sequence of the gene is shown in SEQ ID NO.

21. OcFPN2;1 The nucleotide sequence of the gene is shown in SEQ ID NO.

22.

4. promoting OcFPN1 genes and OcFPN2;1 biological materials that promote gene expression in any one of (1) to (8). (1) improving the nickel accumulation capacity of a plant; (2) preparing a product for improving the nickel accumulation capacity of a plant; (3) improving the tolerance of a plant to nickel stress; (4) preparing a product for improving the tolerance of a plant to nickel stress; (5) creating a nickel hyperaccumulator plant; (6) preparing a product for creating a nickel hyperaccumulator plant; (7) cultivating a plant for remediating nickel-contaminated soil; (8) preparing a product for cultivating a plant for remediating nickel-contaminated soil; the plant is Arabidopsis thaliana; The OcFPN1 The nucleotide sequence of the gene is shown in SEQ ID NO.

21. OcFPN2;1 The nucleotide sequence of the gene is shown in SEQ ID NO.22; the biological material is any one of (1) to (4): (1) a nucleic acid molecule composition consisting of a nucleic acid molecule 1 encoding the OcFPN1 protein and a nucleic acid molecule 2 encoding the OcFPN2;1 protein; (2) an expression cassette containing the nucleic acid molecule composition in (1); (3) a recombinant expression vector containing the expression cassette in (2); (4) a microorganism containing the recombinant expression vector in (3).

5. Use according to any one of claims 3 to 4, characterized in that, The improvement of the nickel accumulation capacity of a plant includes at least one of improving the capacity of a plant to absorb nickel in the underground part, improving the capacity of nickel to be transported from the underground part to the aboveground part of a plant, or improving the capacity of nickel to accumulate in the aboveground part of a plant.

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