Corn nickel transporter and application thereof

Abstract

The invention belongs to the technical field of agricultural biology, relates to application of a protein ZmNPF5.8 with an amino acid sequence as shown in SEQ ID NO: 1 in regulation and control of nickel accumulation in corn kernels and maintenance of storage activity of corn seeds, and can provide gene resources and technical support for cultivation of high-yield and storable corn varieties.

Description

Technical Field

[0001] This invention belongs to the field of agricultural biotechnology, specifically, it relates to a transporter protein ZmNPF5.8 that regulates nickel accumulation in maize kernels and its uses. Background Technology

[0002] As early as 1873, Justus von Liebig, the founder of plant nutrition, discovered a law of plant growth known as Liebig's Law of Minimum. This law states that plant yield depends on the scarcest nutrient, not the total available nutrients, just as the amount of water in a bucket depends on the shortest stave. This law emphasizes the importance of mineral elements in agriculture. However, it is not limited to this, as crop-based foods are a vital source of minerals for human nutrition, and the elemental composition of the edible parts of crops plays a crucial role in human health. Maize is the world's highest-yielding staple crop, with an annual production exceeding 1 billion tons, making it extremely important. Therefore, revealing the genetic basis of elemental balance regulation in maize is of great significance for agricultural progress and human well-being. Although current understanding is limited, research in this area has profound implications.

[0003] Nickel (Ni) was recognized as a beneficial element for crops as early as the 1940s, but it wasn't until the 1980s that it was considered a generally essential element for plants, by which time most other essential elements had been discovered 40 years earlier. Thanks to advancements in analytical chemistry, Eskew et al. (1984) and Brown et al. (1987) established Ni as an essential element for plants from a plant physiological perspective by culturing two or three generations of soybeans and barley in Ni-free, ultrapure broth. However, unlike other essential elements, the importance of nickel in agriculture has never been well recognized, primarily because it was assumed that nickel in most natural soils should be sufficient to meet the needs of most plants. A deeper reason, however, may be that a key gene specifically controlling nickel homeostasis has never been discovered, and nickel deficiency may not have been properly assessed.

[0004] Phytochemical and molecular evidence suggests that some metal chelators, such as nicotianamine (NA), malic acid, and citrate, facilitate the transport of Ni within plants. However, no direct genetic data indicate that loss of function of genes involved in the transport or synthesis of these chelators would lead to severe defects in long-distance Ni transport or distribution. Histidine, a high-affinity nickel chelator, has been shown to enhance nickel uptake in wheat roots, suggesting that histidine may be important for nickel translocation, although the corresponding transporters remain unclear. Without sufficient genetic knowledge, a deeper understanding of the role of nickel in plants is challenging.

[0005] Seed germination is not only a crucial process in the life cycle of all seed plants, but also has significant agronomical implications for crops. The removal of nickel from barley culture medium for three consecutive generations indicates that barley seed viability requires nickel. It has been proposed that urease exists in seeds, and pre-sowing nickel treatment significantly increases urease activity in soybeans. However, in maize, an important food crop in my country, there are currently no reports on nickel absorption, translocation, and distribution. Summary of the Invention

[0006] We conducted a large-scale genome-wide association study (GWAS) and identified a quantitative trait locus (QTL) controlling natural variation in grain Ni content. We further identified the NA efflux transporter gene ZmNPF5.8 as a candidate gene in the maize grain Ni QTL. This gene is specifically expressed in the phloem cells of the maize node, mediating NA secretion into the ectoplasts, thereby promoting Ni transport to the seed. Due to the lack of nickel in the seeds, the ZmNPF5.8 mutant exhibited interrupted seed germination and reduced seed yield, providing the first genetic evidence that nickel is an essential micronutrient for plants. This study confirms that ZmNPF5.8 specifically regulates nickel accumulation in the grain, thereby regulating plant yield and maize grain germination, elucidating its potential utilization value and pathways. Based on this, the present invention includes the following technical solutions.

[0007] The first aspect of this invention provides the use of the protein ZmNPF5.8 (NCBI number Zm00001d044768) or its expression gene ZmNPF5.8, with the amino acid sequence shown in SEQ ID NO:1, in regulating nickel accumulation in plant grains and / or in maintaining the storage activity of plant seeds:

[0008] MSGKTTPTKATLNPF5.8CVLVIVMAGVERFANKGVGSNLVTYLTSVVGMSTAA AAKSVIAWNGVSFMLPLVSAVLADSAHWDRYCTIAASSLLYVLGMVALTTWALLGTRMPRSTLFFPLYLMSIGQGGYQPSLQAFGADQLSIGDDDDDGDGDTEPGASSTPEEKAKVKSMFFRW WYFGMCSGSLLGNSTMSYVQDNLGWGLGFAIPCAVMALSVAAFFCCTPLYKRHRQVQQPKGTGIRPSPSSVFNFKSVLGASRKISLPPSRSSDDNGDAISELELQEKPLKTDEASESESSPDEAAA APGVAKVILGLLPIWAILLVFAVIFQQPMTFFTKQGMLMNHTIGVGVGSGSLVIPPAMLQSSITVSIILLVPMYDRMIVPLTNAVTGGSDGITVLQRIGVGMVLSVVAMVAAALVESRRLRAEP ALLSIFWLLPQYVLLGVSDVFTVVGMQEFFYSQVPASMRTTTGIGLYLSVFGVGGFLGASLITLLEMATARPGNARGWFSDDPREARIDNFYWFLALLCFVSFVVFTHLCKYYNDRSASGR(SEQ ID NO:1).

[0009] The plants mentioned above are preferably monocotyledonous plants, such as grass crops, and can be selected from corn, wheat, rice, soybean, barley, oats, rye and sorghum.

[0010] Preferably, the crop mentioned above is corn.

[0011] In one embodiment, the nucleotide sequence of the expression gene ZmNPF5.8 of the nickel transporter ZmNPF5.8 is SEQ ID NO:2, which consists of a 5'UTR region, a 3'UTR region, introns, and exons (coding region, i.e., CDS).

[0012] In another embodiment, the nucleotide sequence of the expression gene ZmNPF5.8 of the nickel transporter ZmNPF5.8 is SEQ ID NO:3, which is the coding region, i.e., the CDS sequence, in SEQ ID NO:2.

[0013] Preferably, the above-mentioned application refers to increasing the nickel content in corn kernels.

[0014] In the above application method, nickel accumulation in plant grains is increased by overexpressing the protein ZmNPF5.8 or its expression gene ZmNPF5.8 in plants.

[0015] The overexpression of the protein ZmNPF5.8 or its expression gene ZmNPF5.8 can be achieved in the following ways:

[0016] A. The gene ZmNPF5.8 with the nucleotide sequence SEQ ID NO:2 or SEQ ID NO:3 as described above is cloned into a plasmid vector suitable for expression in Agrobacterium to form a recombinant plasmid, namely the ZmNPF5.8 overexpression vector. Plants are then transformed using Agrobacterium-mediated transformation to obtain transgenic plants overexpressing the protein ZmNPF5.8; or

[0017] B. By using gene editing technology, the gene ZmNPF5.8 with the nucleotide sequence SEQ ID NO:2 or SEQ ID NO:3 as described above is cloned into the plant chromosome to obtain a transgenic plant that overexpresses the protein ZmNPF5.8.

[0018] Optionally, the plasmid vector is, for example, a vector used for plant transgenic purposes or a modified vector such as pHB-YFP, pHB-FLAG, pBin19, fluorescent reporter vector pGreenII0800-LUC, pCAMBIA3300, pCAMBIA1301, pCAMBIA2301, pBI121, pTF102.

[0019] For example, the gene editing technology may be selected from the group consisting of: homologous double crossover, TALEN system, CRISPR-Cas9 system, CRISPR-Cpf1 system, CRISPR-Cas12 system, CRISPR-BEST system, and MuGENT.

[0020] A second aspect of the present invention provides a method for identifying maize varieties with high nickel accumulation in their kernels, comprising the following steps:

[0021] Sequencing of the Zm00001d044768 gene in maize, and / or

[0022] The determination was made to determine whether the protein expressed by the maize cells contained a polypeptide with the amino acid sequence shown in SEQ ID NO:1.

[0023] When the detection results show that the maize genome contains the gene ZmNPF5.8 with the nucleotide sequence SEQ ID NO:2 or SEQ ID NO:3, or when the protein expressed by maize cells contains a polypeptide with the amino acid sequence shown in SEQ ID NO:1, it suggests that fishermen have a tendency to accumulate high nickel in the kernels, and this maize variety is considered a candidate for a variety with high nickel accumulation in the kernels.

[0024] When the maize genome does not contain the gene ZmNPF5.8 with the nucleotide sequence SEQ ID NO:2 or SEQ ID NO:3, or when the amplified PCR fragment is a mutant of ZmNPF5.8, or when the protein expressed by maize cells does not contain a polypeptide with the amino acid sequence shown in SEQ ID NO:1, it suggests that maize has a risk of low nickel accumulation in the kernels.

[0025] A third aspect of the present invention provides a kit for performing the above-described identification method, comprising at least the following PCR primers for amplifying the gene ZmNPF5.8:

[0026] Forward primer RT-PCR-NPF5.8-F: CTCTACGTCTTGGGAATGGTA (SEQ ID NO:4),

[0027] Reverse primer RT-PCR-NPF5.8-R: CTGCCGGTGCCGCTTGTACAG (SEQ ID NO:5).

[0028] Preferably, the kit further includes the following PCR primers for detecting the internal reference gene Actin2:

[0029] Forward primer RT-PCR-Actin2-F: CGTCCACCCATAGTGCCATGAG,

[0030] Reverse primer RT-PCR-Actin2-R: ACGGCAAGTAGCACTCAGACAC.

[0031] Furthermore, the kit also includes an instruction manual, which describes the steps and identification criteria for detecting the maize gene Zm00001d044768.

[0032] For example, the instructions can be written on bottles, test tubes and similar objects, boards, or on a separate piece of paper, or on the outside or inside of a container, such as a paper document with an operation demonstration video app download window or a QR code. The instructions can also be in multimedia form, such as a CD, USB flash drive, or cloud storage.

[0033] This invention is the first to discover that the transporter protein ZmNPF5.8 (NCBI ID Zm00001d044768) can positively regulate nickel accumulation in maize kernels, and therefore can be applied to the improvement of plants, especially maize varieties, to enhance seed storage activity. The ZmNPF5.8 gene, as a valuable genetic resource, can be used to breed high-quality new crop varieties. Attached Figure Description

[0034] Figure 1 The GWAS plots of Ni content in maize kernels are shown. A: Manhattan plots of Ni content in kernels 1 and 2 determined by sGWAS, where kernels 1 and 2 are from the 2014 and 2016 CUBIC populations, respectively; the black dashed line represents the cutoff value -log10(p) = 7.0. B: Expression profiles of ZmNPF5.8 in different tissues of the two CUBIC parents. C: Comparison of nickel content in mature kernels of the two haplotypes of CUBIC progeny. D: Relative expression levels of ZmNPF5.8 in the stems of the two haplotypes of CUBIC progeny. E: Comparison of ZmNPF5.8 expression levels in leaves of the two haplotypes of CUBIC progeny.

[0035] Figure 2 The comparison shows the content of nickel and other metal elements in the kernels, the Ni content in the internodes and nodes, and the expression level of the ZmNPF5.8 gene in wild-type maize KN5585, the ZmNPF5.8 gene knockout mutant (crinpf5.8 mutant 1-2), and the ZmNPF5.8 gene overexpression transgenic plants 1-2 (zmnpf5.8-OE mutant 1-2). Among them, AC: bar charts comparing the contents of Ni (Figure A), Zn (Figure B), and Fe (Figure C) in mature kernels of wild-type KN5585 with the ZmNPF5.8 gene knockout mutants crinpf5.8-1 and crinpf5.8-2; D: bar charts comparing the contents of Ni (Figure A), Zn (Figure B), and Fe (Figure C) in mature kernels of wild-type B73 with the EMS-induced mutants npf5.8-1, npf5.8-2, npf5.8-1x, and npf5.8-2F1; E: bar charts comparing the contents of Ni (Figure C) in mature kernels of wild-type B73 with the corresponding background B73; F: bar charts comparing the contents of Ni (Figure C) in KN5585 and the two ZmNPF5.8 overexpression lines zmnpf5.8-OE1 and zmnpf5.8-OE2; F: bar charts comparing the contents of crinpf5.8 mutants crinpf5.8-1 and crinpf5.8-2. G: A bar chart comparing Ni content in different organs (leaf, tassel, stem, and ear) of pf5.8-2 and KN5585 at different growth stages; G: A bar chart comparing Ni content in the growing points of wild-type KN5585 and the zmnpf5.8 mutants crinpf5.8-1 and crinpf5.8-2. Samples of these tissues were collected from the plants after 30 days, and results are expressed as mean ± SDs (n = 6); H: A bar chart comparing Ni content in internodes and nodes of KN5585 and the zmnpf5.8 mutants crinpf5.8-1 and crinpf5.8-2. Samples of these tissues were collected from the plants 30 days after pollination. Values ​​are expressed as mean + standard deviation, and significance is indicated by a t-test (*P < 0.05, ***P < 0.001). Figure 2 This study demonstrated that ZmNPF5.8 is the causal site for natural variation in Ni content in maize kernels, and Figure E further proved that overexpression of the ZmNPF5.8 gene can increase Ni content in maize kernels.

[0036] Figure 3 This study demonstrates the role of ZmNPF5.8 as a NA (nicotianamine) efflux transporter. A: Representative image of leaf epidermal cells expressing pUbi::ZmNPF5.8-eYFP; after 30 min of BFA treatment in seedlings, ZmNPF5.8-eYFP aggregated to form BFA bodies. B: Colocalization of ZmNPF5.8-GFP and AtNAET2-mCherry in leaf epidermal cells of *N. benthamiana*. C: Colocalization of ZmNPF5.8 and NA in synaptic vesicles; immunostaining of leaf epidermal cells of the transgenic line expressing pUbi::ZmNPF5.8-eYFP with anti-NA antibody shows fluorescence of the secondary antibody (purple) and green fluorescent protein (green). D: Identification of ZmNPF5.8 as a NA efflux transporter using a yeast system; the introduction of NA pPR3-... The AhNAS3 or pPR3 vector was introduced into yeast strain NMY51 containing different genes in the expression vector pBT3. Transformants were cultured in YPD medium, and the NA content in the secretory medium and the NA content in the cells of yeast with different structures were detected by UPLC-MS. E: Representative image of GUS-stained stem expressing pZmNPF5.8::GUS against KN5585 background; F: Representative image of GUS-stained leaf expressing pZmNPF5.8::GUS against KN5585 background; G: Representative image of GUS-stained leaf sheath tissue expressing pZmNPF5.8::GUS against KN5585 background; H: Representative image of GUS-stained scion stem expressing pZmNPF5.8::GUS against KN5585 background. Figure 3 This indicates that ZmNPF5.8 functions as a NA (nicotianamine) efflux transporter, meaning that ZmNPF5.8 is a transport protein.

[0037] Figure 4The growth phenotypes of wild-type maize KN5585 and the ZmNPF5.8 gene knockout mutants crinpf5.8-1 and crinpf5.8-2 are compared, as are the growth phenotypes of wild-type B73 and the EMS-induced mutants npf5.8-1 and npf5.8-2. Among them, AB: comparison of the whole plant phenotype of KN5585 grown in the nursery with the mutants crinpf5.8-1 and crinpf5.8-2 (Figure A); comparison of the whole plant phenotype of wild-type B73 grown in the nursery with the induced mutants npf5.8-1 and npf5.8-2 (Figure B); CD: bar chart comparing the plant height of wild-type KN5585 with the mutants crinpf5.8-1 and crinpf5.8-2 (Figure C); comparison of the plant height of wild-type B73 with the induced mutants npf5.8-1 and npf5.8-2 (Figure C). Figure D); Figures EF: Comparison of ear type between wild-type KN5585 and mutants crinpf5.8-1 and crinpf5.8-2 in field growth (Figure E); Comparison of ear type between wild-type B73 and mutants npf5.8-1 and npf5.8-2 (Figure F); Figures GH: Bar charts comparing ear weight between wild-type KN5585 and mutants crinpf5.8-1 and crinpf5.8-2 in field growth (Figure G); Bar charts comparing ear weight between wild-type B73 and mutants npf5.8-1 and npf5.8-2 (Figure H). Values ​​are the mean plus standard deviation. Significance is indicated by a t-test (*P<0.05, ***P<0.001). Figure 4 This indicates that nickel is crucial for yield traits and seed germination.

[0038] Figure 5This paper presents a comparison of seed germination phenology between wild-type maize KN5585 and the ZmNPF5.8 gene knockout mutants crinpf5.8-1 and crinpf5.8-2. Specifically: A: Seed germination phenology of wild-type KN5585 and mutants crinpf5.8-1 and crinpf5.8-2; B: Bar graph comparing the taproot length of germinated seeds from mutants crinpf5.8-1 and crinpf5.8-2 with wild-type KN5585; C: Comparison of seed germination of ZmNPF5.8 gene knockout mutants crinpf5.8-1 and wild-type KN5585 under different Ni complement levels for 24 hours; D: Different... Comparison of seed germination phenology of storage time mutants crinpf5.8-1 and crinpf5.8-2 with wild-type KN5585; EF: Bar chart comparing the relative contents of arginine (Figure E) and glutamine (Figure F) in germinating seeds of crinpf5.8-1 mutant and wild-type KN5585; G: Schematic diagram of urease metabolic pathway; H: Urease activity during seed germination of mutants crinpf5.8-1 and crinpf5.8-2 with wild-type KN5585. Detailed Implementation

[0040] In this study, our research group discovered for the first time that the ZmNPF5.8 gene in the maize genome can regulate nickel accumulation in maize kernels. Comparison between wild-type maize KN5585 and B73 and maize mutants with the ZmNPF5.8 gene knockout revealed that knockout or mutation of the ZmNPF5.8 gene significantly reduced nickel (Ni) content in maize kernels, and the mutant plants exhibited poorer growth phenotypes, suggesting that this gene controls the redistribution of nickel to the kernels. Knockout mutant kernels had a lower germination rate under the same storage conditions than wild-type kernels, while exogenous nickel treatment could salvage the germination rate to some extent, indicating that nickel deficiency caused by the loss of gene function leads to decreased kernel viability. From another perspective, the ZmNPF5.8 gene plays a maintaining and / or enhancing role in Ni accumulation and growth of maize kernels.

[0041] Based on this discovery, the present invention can use the expression of the gene ZmNPF5.8 (Zm00001d044768) as an indicator to identify maize varieties with nickel accumulation in kernels. The identification method involves sequencing the maize gene Zm00001d044768 and / or determining whether the protein expressed by the maize cells contains the polypeptide NPF5.8 with the amino acid sequence shown in SEQ ID NO:1. If the maize genome contains the gene ZmNPF5.8 with the nucleotide sequence SEQ ID NO:2 or SEQ ID NO:3, and / or the protein expressed by the maize cells contains the polypeptide NPF5.8 with the amino acid sequence shown in SEQ ID NO:1, i.e., NPF5.8 is present and has not been mutated or inactivated, it suggests that the maize has a tendency to accumulate nickel in kernels, and this maize variety can be considered a candidate for a variety with nickel accumulation in kernels and good seed storage.

[0042] As used in this article, the term "wild-type" refers to native plants, such as maize, that have not undergone genetic modification or mutagenesis.

[0043] Correspondingly, the terms "(plant) mutant", "transgenic plant" and "genetically engineered plant" in this article have the same meaning, all referring to plants that have been genetically modified or artificially mutated from wild-type plants.

[0044] 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).

[0045] As used herein, the terms “(grain nickel content) increase,” “enhancement,” or “increase” can mean an increase of at least 10% compared to a reference level (e.g., wild-type maize / initial maize), such as an increase 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 100%, or any increase between 10% and 100%, or an increase 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.

[0046] In this document, for the sake of simplicity, the name of a protein, such as the transporter protein ZmNPF5.8 (abbreviated as NPF5.8), and its encoding gene (DNA) name ZmNPF5.8 are sometimes used interchangeably. Those skilled in the art should understand that they represent different types of 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 category of regulating nickel accumulation, NPF5.8 refers to the protein; when used as a gene description, it refers to the gene encoding that protein.

[0047] On the other hand, given that the gene ZmNPF5.8 with a normal sequence or its encoded protein NPF5.8 (Zm00001d044768) has a positive role in regulating nickel accumulation in grains, it is possible to try to breed plant varieties with high nickel accumulation in grains by overexpressing the protein ZmNPF5.8 with the amino acid sequence shown in SEQ ID NO:1 in plants such as maize, thereby constructing the desired grain nickel accumulation varieties and genetically engineered plants with seeds that are resistant to storage.

[0048] The advantage of the aforementioned gene identification scheme lies in the fact that it is possible to pre-assess, solely in the laboratory, whether candidate plant varieties have the potential for grain nickel accumulation and seed storage durability. Since the entire life cycle of crops such as corn is typically one year, examining their biological traits and phenotypes through field cultivation would normally require a significant amount of time and resources, including substantial land and labor costs. In contrast, the gene identification scheme can be completed in the laboratory, allowing for gene sequencing in a short period, such as on seedlings within a few weeks, or even just on seeds. This significantly improves efficiency and substantially reduces time, space, and labor costs, resulting in substantial economic benefits.

[0049] The construction of such ZmNPF5.8 overexpressing genetically engineered plants can be achieved using traditional Agrobacterium-mediated transformation of recombinant plasmids or gene editing technology.

[0050] In a specific implementation, an NPF5.8 (Zm00001d044768) expression cassette and nucleic acid construct, or expression construct, is constructed based on the gene ZmNPF5.8 with nucleotide sequence SEQ ID NO:2 or SEQ ID NO:3. Then, an NPF5.8 expression plasmid is constructed, and finally, ZmNPF5.8 as a foreign gene is expressed in wild plants / original plants.

[0051] The coding sequence of the polypeptide ZmNPF5.8 of the present invention, or a fragment thereof, can generally be obtained by PCR amplification, recombination, or artificial synthesis. For PCR amplification, conventional techniques can be used to amplify the ZmNPF5.8 gene from genomic DNA, and primers can be designed based on the nucleotide sequence disclosed in the present invention, especially the open reading frame sequence.

[0052] As used herein, the term "expression cassette," "gene expression cassette," or "nucleic acid construct" refers to a gene expression system containing all the necessary elements required to express the target polypeptide NPF5.8, typically including the following elements: a promoter, a gene sequence encoding the polypeptide, and a terminator; additionally, it may optionally include a signal peptide encoding sequence, etc.; these elements are operatively linked.

[0053] As used herein, an "expression construct" or "expression building block" refers to a recombinant DNA molecule containing the intended nucleic acid coding sequence SEQ ID NO:2 or 3, which may contain one or more gene expression cassettes. The "construct" is typically contained within an expression vector (plasmid vector).

[0054] As used herein, “operationally linked” or “operationally connected” refers to a functional spatial arrangement of two or more nucleic acid regions or nucleic acid sequences. For example, a promoter region is placed at a specific position relative to the target gene nucleic acid sequence SEQ ID NO:2 or 3, such that transcription of the nucleic acid sequence is guided by the promoter region, thereby the promoter region is “operationally linked” to the nucleic acid sequence.

[0055] The nucleic acid constructs described in this invention can be manipulated in various ways to ensure the expression of the polypeptide or protein ZmNPF5.8. The nucleic acid constructs can be manipulated according to the different expression vectors or requirements before insertion into the vector. Techniques for altering polynucleotide sequences using recombinant DNA methods are known in the art.

[0056] In some embodiments, the nucleic acid construct is a vector. The vector can be a cloning vector, an expression vector, or a gene knock-in vector. The nucleic acid sequence SEQ ID NO:2 or 3 of the present invention can be cloned into many types of vectors, such as plasmids, phage particles, phage derivatives, animal viruses, and granules. Cloning vectors can be used to provide the coding sequence of the protein or polypeptide of the present invention. Expression vectors can be provided to cells in the form of bacterial or viral vectors. Expression of the ZmNPF5.8 gene of the present invention is typically achieved by operably linking the nucleic acid sequence SEQ ID NO:2 or 3 of the present invention to a promoter and incorporating the construct into an expression vector. This vector is suitable for replication and integration into eukaryotic cells. Typical expression vectors contain expression control sequences that can be used to regulate the expression of the desired nucleic acid sequence.

[0057] Gene knock-in vectors can be used to integrate the polynucleotide sequence SEQ ID NO:2 or 3 described herein into a region of interest in the host genome. Typically, in addition to the polynucleotide sequence described herein, gene knock-in vectors may also contain 5' and 3' homologous arms required for genomic homologous recombination. In some embodiments, the nucleic acid constructs described herein contain 5' homologous arms, the polynucleotide sequence described herein, and 3' homologous arms. When using gene knock-in vectors, CRISPR / Cas9 technology can be used simultaneously to homologously recombine the polynucleotide sequence into the site of interest. CRISPR / Cas9 technology guides the Cas9 nuclease to modify the genome at the insertion site by designing guide RNAs targeting the target gene, resulting in increased homologous recombination efficiency in the modified region, thus homologously recombinating the target fragment SEQ ID NO:2 or 3 contained in the gene knock-in vector into the target site. The steps of CRISPR / Cas9 technology and the reagents used, such as the Cas9 nuclease, are well known in the art.

[0058] Methods well known to those skilled in the art can be used to construct nucleic acid constructs. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, and in vivo recombination techniques. The DNA sequence can be efficiently ligated to an appropriate promoter in the expression vector to direct mRNA synthesis. Representative examples of these promoters include: the lac or trp promoter of *E. coli*; the PL promoter of *λ* phage; eukaryotic promoters including the CMV immediate early promoter, the HSV thymidine kinase promoter, early and late SV40 promoters, LTRs of retroviruses, and other known promoters that control gene expression in prokaryotic or eukaryotic cells or their viruses. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator. Furthermore, the expression vector preferably contains one or more selective marker genes to provide phenotypic traits for selecting host cells for transformation, such as dihydrofolate reductase, neomycin resistance, and green fluorescent protein (GFP) for eukaryotic cell culture, or tetracycline, ampicillin resistance, or chloramphenicol for *E. coli*, *Agrobacterium*, etc.

[0059] When the polynucleotides of this invention are expressed in higher eukaryotic cells, the insertion of an enhancer sequence into the vector will enhance transcription. Enhancers are cis-acting factors of DNA, typically approximately 10 to 300 base pairs, that act on the promoter to enhance gene transcription. Examples include the SV40 enhancer (100 to 270 base pairs) located late on the replication origin side, the polyoma enhancer located late on the replication origin side, and adenovirus enhancers.

[0060] Vectors containing appropriate DNA sequences and appropriate promoters or control sequences can be used to transform appropriate host cells so that they can express proteins.

[0061] When constructing transgenic plants using the traditional Agrobacterium-mediated transformation method, the methods for constructing transgenic plants include:

[0062] 1) Provide Agrobacterium carrying an expression vector, wherein the expression vector contains the coding sequence of the polypeptide ZmNPF5.8;

[0063] 2) Contact plant cells, tissues, or organs with Agrobacterium in step 1) to transfer the coding sequence into the plant cells and integrate it into the chromosomes of the plant cells;

[0064] 3) Select plant cells or tissues into which the coding sequence has been introduced; and

[0065] 4) Regenerate plants from the plant cells or tissues in step 3).

[0066] The method described herein can be used to construct transgenic plants with high nickel accumulation in the grains and / or good seed storage properties.

[0067] The present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0068] Example

[0069] 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.

[0070] In the embodiments described herein, unless otherwise specified, the temperature generally refers to room temperature (15-30°C).

[0071] The molecular biology experiments in this embodiment, including plasmid construction, enzyme digestion, competent cell preparation, and transformation, were mainly conducted in accordance with *Molecular Cloning: A Laboratory Manual* (3rd Edition), edited by J. Sambrook and DW. Russell (USA), translated by Huang Peitang et al., Science Press, Beijing, 2002. For example, the methods for competent cell transformation and competent cell preparation were both performed according to Chapter 1, page 96 of *Molecular Cloning: A Laboratory Manual* (3rd Edition). Specific experimental conditions could be determined through simple experiments if necessary.

[0072] 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.

[0073] The primer synthesis and gene sequencing in this embodiment were outsourced to Sangon Biotech (Shanghai) Co., Ltd.

[0074] Example 1: Maize Genome-Wide Association Analysis Method

[0075] In the CUBIC population of maize, we used the ionotype content of kernels harvested from the Hainan experimental base of the Shanghai Institute of Plant Physiology and Ecology, Chinese Academy of Sciences in 2014 and 2016, after Log2 transformation by ICP-MS, as the phenotype. We used 11.8 million high-quality SNPs (minimum allele frequency MAF ≥ 0.02) obtained from previous analyses by the Yan Jianbing research group at Huazhong Agricultural University as genotypes. Single-marker association analysis (sGWAS) was performed using a mixed linear model in Tassel 3.0 software. To control for the effects of rare alleles while preserving their potential roles, we used 11.8 million SNPs with MAF ≥ 2% as genotype data. SNP genotype and population structure were treated as fixed effects, while the kinship matrix was treated as a random effect. The threshold was set to 1 / N, where N is the total number of SNP markers actually used for each trait. SNPs with a p-value less than the set threshold were considered significant. The SNP with the lowest p-value was the most significant peak SNP. The physical interval covered by each SNP locus was defined as the interval of that sQTL.

[0076] Figure 1 The process and results of the maize genome-wide association analysis in this embodiment are shown.

[0077] The investigation of metal ion content revealed that nickel (Ni) ion content has a significant impact on the biological traits of maize. See the GWAS diagram of Ni content in maize kernels. Figure 1 Figure A shows a Manhattan plot of Ni content in kernel 1 and kernel 2 determined by sGWAS, where kernel 1 and kernel 2 represent kernels from the 2014 and 2016 CUBIC populations, respectively. Figure B shows the expression profile of gene ZmNPF5.8 in different tissues of the two CUBIC parents. Figure C shows the Ni content in maize kernels of CUBIC progeny. Figures D and E show the expression of the ZmNPF5.8 gene in kernels and leaves of CUBIC progeny. The results in Figures C and D indicate a positive correlation between gene ZmNPF5.8 expression and Ni content in maize kernels.

[0078] The nucleotide sequence of the gene ZmNPF5.8 is SEQ ID NO:2, which consists of a 5'UTR region, a 3'UTR region, introns, and exons (coding region, i.e., CDS). The nucleotide sequence of the exons (CDS) is SEQ ID NO:3, which encodes a protein ZmNPF5.8 (abbreviated as NPF5.8) with an amino acid sequence as shown in SEQ ID NO:1.

[0079] Example 2: Subcellular localization of ZmNPF5.8

[0080] Subcellular localization of the ZmNPF5.8 gene was performed using the following steps.

[0081] 1. Transient expression of ZmNPF5.8 in tobacco: Based on the cDNA sequence of ZmNPF5.8, PCR amplification was performed using the high-fidelity enzyme KODplus (TOYOBO) with the cDNA as a template. Simultaneously, corresponding primers were designed to amplify the GFP fragment. ZmNPF5.8 was fused to the GFP fragment at the N-terminus using overlap PCR. The successfully ligated PCR product was recovered by electrophoresis and cloned into the transient transgenic vector pA7. Positive clones were sequenced to verify their correctness and then stored.

[0082] 2. The transient expression vector was transformed into Agrobacterium, and positive clones were screened using cannabidiol medium. Single clones were picked and shaken in a shaker at 28°C until they turned orange. The bacterial cells were collected, suspended in Agrobacterium injection solution, and injected into the underside of Nicotiana benthamiana leaves. After 48 hours, the cells were observed using a Leica SP8 laser confocal microscope.

[0083] Figure 3 The AC diagram shows the subcellular localization of the ZmNPF5.8 gene in this embodiment.

[0084] Example 3: Yeast complementation assay to verify that ZmNPF5.8 transports the Cu-NA complex into yeast cells. The transporter function of ZmNPF5.8 was investigated through the following steps.

[0085] 1. Construction of ZmNPF5.8 yeast expression vector

[0086] Based on the cDNA sequence of ZmNPF5.8, specific primers with the stop codon removed were designed. PCR amplification was performed using the cDNA as a template with the high-fidelity enzyme KODplus (TOYOBO). The PCR products were recovered by electrophoresis and cloned into the yeast expression vector pDR196. Positive clones were sequenced to verify their correctness and then stored.

[0087] 2. Yeast Conversion and Absorption Test

[0088] Transformants containing different genes were introduced into the pPR3-AhNAS3 or pPR3 empty vector into the expression vector pBT3 of yeast strain NMY51, and cultured in YPD medium. The NA content in the secretory medium and the NA content in the cells of yeast strains with different structures were detected by UPLC-MS.

[0089] See Figure 3 The experimental results showed that the transporter protein ZmNPF5.8 transported the Cu-NA complex into yeast cells.

[0090] Example 4: Construction of ZmNPF5.8 gene knockout mutants crinpf5.8-1 and crinpf5.8-2

[0091] The ZmNPF5.8 gene knockout mutant was constructed and cultured by Weimi Biotechnology (Changzhou) Co., Ltd., ultimately yielding two ZmNPF5.8 gene knockout mutant plants, crinpf5.8-1 and crinpf5.8-2. [Participants...] Figure 2 .

[0092] Example 5: Construction of transgenic maize zmnpf5.8-OE1 and zmnpf5.8-OE2 overexpressing the ZmNPF5.8 gene

[0093] Transgenic maize overexpressing the ZmNPF5.8 gene was constructed and cultivated by Weimi Biotechnology (Changzhou) Co., Ltd., ultimately yielding two transgenic plants, zmnpf5.8-OE1 and zmnpf5.8-OE2, overexpressing the ZmNPF5.8 gene. (Participants...) Figure 2 .

[0094] Example 6: Obtaining EMS-induced mutants npf5.8-1, npf5.8-2, npf5.8-1x, and npf5.8-2F1

[0095] The EMS-induced mutant was purchased from the maize EMS mutant library of Qilu Normal University. Four EMS-induced mutants were ultimately obtained: npf5.8-1, npf5.8-2, npf5.8-1x, and npf5.8-2F1. [The text abruptly ends here, likely due to an incomplete sentence or missing information.] Figure 2 .

[0096] Example 7: ICP-MS Detection of Nickel Ion Content

[0097] Using ICP-MS, the levels of Ni, Zn, and Fe ions were detected in the kernels and leaves of the following maize plants: wild-type maize KN5585 and B73, including two haplotype CUBIC progeny; two ZmNPF5.8 gene knockout mutants, crinpf5.8-1 and crinpf5.8-2; two transgenic plants overexpressing the ZmNPF5.8 gene, zmnpf5.8-OE1 and zmnpf5.8-OE2; and four EMS-induced mutants, npf5.8-1, npf5.8-2, npf5.8-1x, and npf5.8-2F1. The steps included:

[0098] 1. Sample selection and processing

[0099] For samples of grains from the population, three mature female ears of uniform growth were selected from each offspring. Three seeds from each ear were taken, mixed together, and ground into a uniform dry powder. Approximately 5 mg of this powder was weighed as the test sample. For different parts of mature grains from mutants and wild types, the dry weight was measured after dissection with a plastic knife. The entire embryo or endosperm of each seed was considered as one sample. For different parts of grains during development from mutants and wild types, the wet weight was measured after dissection with a plastic knife. The entire embryo or endosperm of each seed was considered as one sample.

[0100] 2. Place the corn kernel sample into a glass test tube and dry it overnight in a 65℃ oven. The next day, remove the glass test tube and cool it to room temperature.

[0101] 3. Open the fume hood, add 2 ml of concentrated nitric acid containing indium (In) to each glass test tube, place them in a graphite furnace, and treat at 115°C for 4 hours. Add ultrapure water to a final volume of 10 ml (this step requires accounting for evaporation during heating), and gently mix well. Transfer 1 ml of the mixture to a new set of glass test tubes.

[0102] 4. Add 1 ml of concentrated nitric acid containing In, place it in a graphite furnace, and treat it at 115°C for 4 hours.

[0103] 5. Add ultrapure water to bring the volume to 10 ml (this step requires consideration of the amount evaporated by heating), mix gently and evenly, and add 1.7 ml of the mixture to the specific sample plate in sequence. The ion content in the sample can be determined using the ICP-MS (NexION 300D; PerkinElmer) instrument of this group.

[0104] See results Figure 2 Figure A shows that compared with wild-type KN5585, the Ni content in the kernels of the knockout mutants crinpf5.8-1 and crinpf5.8-2 decreased by approximately 72-74%; Figure B shows that compared with wild-type KN5585, the Zn content in the kernels of the knockout mutants crinpf5.8-1 and crinpf5.8-2 decreased by approximately 20-22%; Figure C shows that the Fe content in the kernels of wild-type KN5585 is not significantly different from that of the knockout mutants crinpf5.8-1 and crinpf5.8-2, indicating that ZmNPF5.8 has a significant impact on Ni accumulation in kernels. Figure D shows that compared with wild-type KN5585, the Ni content in the kernels of the EMS-induced mutants npf5.8-1, npf5.8-2, and npf5.8-1x decreased by approximately 66-70%. Figure E shows that compared with wild-type KN5585, the Ni content in the kernels of the overexpressing transgenic plant zmnpf5.8-OE1 increased by 7.5 times, while the Ni content in the kernels of another overexpressing plant zmnpf5.8-OE2 increased by 1.2 times. This indicates that ZmNPF5.8 positively regulates Ni accumulation in maize kernels, and overexpression of the ZmNPF5.8 gene can increase the Ni content in maize kernels. Figure F shows that the Ni content varies in different organs (leaf, bract leaf, tassel, stem, and ear) of wild-type KN5585 and knockout mutants crinpf5.8-1 and crinpf5.8-2 at different growth stages. Figure G shows that compared with wild-type KN5585, the Ni content in the growth points of knockout mutants crinpf5.8-1 and crinpf5.8-2 decreased by 65-68%. Figure H shows that compared with wild-type KN5585, the Ni content in the internodes of knockout mutants crinpf5.8-1 and crinpf5.8-2 is not significantly different, but the Ni content in the nodes of mutants crinpf5.8-1 and crinpf5.8-2 is higher than that of wild-type KN5585, further indicating that ZmNPF5.8 positively regulates Ni accumulation in maize kernels.

[0105] Example 8: GUS staining

[0106] The function of ZmNPF5.8 was investigated using GUS staining, including the following steps:

[0107] 1. Place the material in 90% acetone pre-chilled on ice. Incubate at room temperature for 20 minutes.

[0108] 2. Discard the acetone and wash twice with GUS-buffer.

[0109] 3. Add GUS staining solution and vacuum on ice for 1-2 hours.

[0110] 4. Stain at 37°C in the dark for several hours or overnight until a signal appears.

[0111] 5. After staining, discard the staining solution, add 70% ethanol to stop the reaction and decolorize, and observe under a microscope.

[0112] The results are as follows Figure 3 As shown. Figure 3 The comparison results of AH indicate that ZmNPF5.8 functions as a NA (nicotianamine) efflux transporter, meaning that ZmNPF5.8 is a transport protein.

[0113] Comparison of seed germination phenotypes, arginine and glutamine contents, and urease metabolic pathways between wild-type maize KN5585 and the ZmNPF5.8 gene knockout mutants crinpf5.8-1 and crinpf5.8-2, as well as analysis of these parameters, showed that... Figure 5 Further analysis showed that ZmNPF5.8 has the effect of promoting Ni accumulation in maize kernels.

[0114] The embodiments described above are merely preferred embodiments for fully illustrating the present invention, and the scope of protection of the present invention is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art based on the present invention are all within the scope of protection of the present invention.

Claims

1. The application of the protein ZmNPF5.8 (NCBI number Zm00001d044768) with the amino acid sequence shown in SEQ ID NO:1 or its expression gene ZmNPF5.8 in regulating nickel accumulation in plant grains and / or in maintaining the storage activity of plant seeds.

2. The application as described in claim 1, characterized in that, The plants in question are grass crops, selected from corn, rice, wheat, soybean, barley, oats, rye, and sorghum.

3. The application as described in claim 2, characterized in that, The crop in question is corn.

4. The application as described in claim 1, characterized in that, The nucleotide sequence of the gene expressing the protein ZmNPF5.8 is SEQ ID NO:2, which consists of a 5'UTR region, a 3'UTR region, introns, and exons (coding region, i.e., CDS).

5. The application as described in claim 1, characterized in that, The nucleotide sequence of the gene expressing the protein ZmNPF5.8 is SEQ ID NO:3, which is the coding region, i.e., the CDS sequence, in SEQ ID NO:

2.

6. The application as described in claim 1, characterized in that, Used to increase the nickel content in corn kernels.

7. The application as described in claim 1, characterized in that, Increased nickel accumulation in plant grains can be achieved by overexpressing the protein ZmNPF5.8 or its expression gene ZmNPF5.8 in plants.

8. The application as described in claim 6, characterized in that, The overexpressed protein ZmNPF5.8 or its expression gene ZmNPF5.8 is achieved through the following method: A. The gene ZmNPF5.8 with the nucleotide sequence SEQ ID NO:2 or SEQ ID NO:3 as described in claim 4 or 5 is cloned into a plasmid vector suitable for expression in Agrobacterium to form a recombinant plasmid, namely a ZmNPF5.8 overexpression vector, and plants are transformed by Agrobacterium-mediated transformation to obtain transgenic plants that overexpress the protein ZmNPF5.

8. or B. By using gene editing technology, the gene ZmNPF5.8 with the nucleotide sequence SEQ ID NO:2 or SEQ ID NO:3 as described in claim 4 or 5 is cloned into the plant chromosome to obtain a transgenic plant that overexpresses the protein ZmNPF5.

8.

9. A method for identifying maize varieties with high nickel accumulation in kernels, characterized in that, Includes the following steps: Sequencing of the Zm00001d044768 gene in maize, and / or The determination was made to determine whether the protein expressed by the maize cells contained a polypeptide with the amino acid sequence shown in SEQ ID NO:

1. When the detection results show that the maize genome contains the gene ZmNPF5.8 with the nucleotide sequence SEQ ID NO:2 or SEQ ID NO:3, or when the protein expressed by maize cells contains a polypeptide with the amino acid sequence shown in SEQ ID NO:1, it suggests that fishermen have a tendency to accumulate high nickel in the kernels, and this maize variety is considered a candidate for a variety with high nickel accumulation in the kernels. When the maize genome does not contain the gene ZmNPF5.8 with the nucleotide sequence SEQ ID NO:2 or SEQ ID NO:3, or when the amplified PCR fragment is a mutant of ZmNPF5.8, or when the protein expressed by maize cells does not contain a polypeptide with the amino acid sequence shown in SEQ ID NO:1, it suggests that maize has a risk of low nickel accumulation in the kernels.

10. A kit for carrying out the method as described in claim 9, characterized in that, The following PCR primers are included for amplifying the gene ZmNPF5.8: Forward primer RT-PCR-NPF5.8-F: CTCTACGTCTTGGGAATGGTA (SEQ ID NO:4), Reverse primer RT-PCR-NPF5.8-R: CTGCCGGTGCCGCTTGTACAG (SEQ ID NO:5).

Images