CN104212815B - Maize zinc-iron regulation transporter ZmIRT1 gene and application thereof - Google Patents

Abstract

The invention discloses a ZmIRT1 gene of a maize zinc and iron regulation transporter and an application thereof. The present invention isolates the ZmIRT1 gene of the zinc-iron regulated transporter from maize, and its cDNA sequence is shown in SEQ ID No.1. Subcellular localization shows that the zinc-iron-regulated transporter is localized in the plasma membrane and endoplasmic reticulum of cells, and plays roles in absorbing, transporting and storing zinc-iron and participating in the detoxification of zinc-iron ions in plant cells. Real-time quantitative RT-PCR expression analysis found that the expression of ZmIRT1 was up-regulated in the roots and shoots of zinc-deficient or iron-deficient plants, and the expression was significantly up-regulated in the later stage of embryo development, which played an important role in regulating embryo development. Yeast complementation experiments showed that ZmIRT1 gene has the ability of zinc-iron complementation. The ZmIRT1 gene isolated by the invention has important application prospects for regulating the ability of plants to absorb, transport and store zinc and iron, promoting the development of embryos, increasing the content of zinc and iron in grain crop grains, and the like.

Description

ZmIRT1 gene of maize zinc and iron regulated transporter and its application

technical field

The present invention relates to metal ion transporter genes isolated from plants, in particular to the regulatory transporter ZmIRT1 gene isolated from corn and related to the absorption, transport or storage of zinc and iron. The present invention further relates to the regulatory transporter ZmIRT1 gene in regulating The application in the ability of plants to absorb, transport or store zinc or iron, detoxify excessive zinc and iron to plants, and increase the content of zinc and iron in grain crop seeds belongs to the field of isolation and application of plant metal ion-regulated transporter genes.

Background technique

Zinc and iron are essential trace elements for organisms and play an important role in the growth and development of plants (Wintz H, Fox T, Wu YY, et al. Expression profiles of Arabidopsis thaliana inorganic deficiencies reveal novel transporters involved in metalhomeostasis.The Journal of biological chemistry 2003, 278(48): 47644-47653.). Zinc is a structural cofactor for more than 300 enzymes and important proteins in organisms (Haydon MJ, Cobbett CS. A novel major facilitator superfamily protein at the tonoplast influences zinctolerance and accumulation in Arabidopsis. Plant physiology 2007,143(4):1705-1719. ). Zinc not only participates in various metabolisms of the body, but also plays an important role in physiological functions such as biofilm stability and gene expression regulation (Mathews WR, Wang F, Eide DJ, et al.Drosophila fear of intimacyencodes a Zrt/IRT-like protein (ZIP) family zinc transporter functionally related to mammalian ZIP proteins. The Journal of biological chemistry 2005,280(1):787-795.). Zinc deficiency can lead to oxidative damage to chlorophyll, lipids, proteins, and plasma membranes. Properly increasing the zinc content in plants can increase crop yields, while excessive accumulation of zinc ions in plants can be toxic to plants.

Iron plays an important role in the process of cellular respiration, photosynthesis and metalloprotein catalysis, and is an important electron carrier. Therefore, iron element has an irreplaceable function in the life activities of prokaryotic and eukaryotic organisms. In addition, too high redox potential of Fe ³⁺ /Fe ²⁺ in cells will lead to the production of superoxide compounds, causing damage to cells (Briat JF, Lebrun M. Plant responses to metal toxicity. Comptes rendus de l'Academie dessciences Serie III, Sciences de la vie 1999, 322(1):43-54.). Therefore, it is crucial to strictly control the balance of metal ions in plants.

There are three main types of proteins involved in zinc and iron absorption, all of which exist in the form of protein families, including: ZIP, namely zinc-regulated transporter (Zinc-regulated transporter, ZRT) and iron-regulated transporter (Iron-regulated transporter, IRT). Yeast functional complementation experiments showed that ZIP family genes can transport various metal ions including Zn ²⁺ , Fe ²⁺ , Cu ²⁺ , and Cd ²⁺ (Colangelo EP, Guerinot ML. Put the metal to the petal: metaluptake and transport throughout plants. Current opinion in plant biology 2006,9(3):322-330.). ZIP generally consists of 309-476 amino acid residues, has 8 potential transmembrane domains and similar topology, there is a long variable region between the 3rd and 4th transmembrane domain, and the variable region is located in the cell , its C and N terminals are located extracellularly, and this region is rich in histidine residues, which may be related to the binding and transport of metals (Guerinot ML.The ZIP family of metal transporters.Biochim Biophys Acta 2000,1465(1-2): 190-198.).

At present, ZIP genes have been identified in Arabidopsis, rice, Medicago truncatula, soybean, wild-type emmer, grape and other plants, and their functions have been studied. Sixteen ZIP family genes were found in Arabidopsis, and AtIRT1 is the first ZIP functional gene isolated by yeast complementation experiment, which is mainly expressed in roots, and overexpression of this gene can lead to excessive accumulation of nickel (Eide D, Broderius M, Fett J, et al. A novel iron-regulated metal transporter from plants identified by functional expression in yeast. Proceedings of the National Academy of Sciences of the United States of America 1996,93(11):5624-5628; Henriques R, Jasik J, Klein M, et al. Knock-out of Arabidopsis metaltransporter gene IRT1 results in iron deficiency accompanied by cell differentiation defects. Plant molecular biology 2002,50(4-5):587-597; VarottoC, Maiwald D, Pesaresi P, et al. al. The metal ion transporter IRT1 is necessary foriron homeostasis and efficient photosynthesis in Arabidopsis thaliana. The Plant journal: for cell and molecular biology 2002,31(5):589-599; Vert G, GrotzN, Dedaldecamp F, et al. IRT1, an Arabidopsis transporter essential for ironuptake from the soil and for plant growth. Plant Cell 2002,14(6):1223-1233; Nishida S, Tsuzuki C, Kato A, et al. AtIRT1, the primary iron uptake transporter in the root, mediates excess Nickel accumu lation in Arabidopsisthaliana. Plant & cell physiology 2011,52(8):1433-1442.). AtIRT2 is mainly expressed in roots and localized in vesicles. It is speculated that it has the function of detoxifying excess metal elements in cells (Vert G, Briat JF, Curie C. Arabidopsis IRT2 gene encodes a root-periphery iron transporter. The Plantjournal: for cell and molecular biology 2001, 26(2):181-189; Vert G, Barberon M, Zelazny E, et al. Arabidopsis IRT2cooperates with the high-affinity iron uptakesystem to maintain iron homeostasis in root epidermal cells. Planta 2009,229(6):1171-1179.14 ,15). AtIRT3 can complement zinc and iron transport double mutants, and overexpression of AtIRT3 will cause zinc to accumulate in the upper part and iron in the underground part (Lin YF, Liang HM, Yang SY, et al. Arabidopsis IRT3 is a zinc-regulated and plasma membrane localized zinc /iron transporter. The Newphytologist 2009, 182(2):392-404.). Expression analysis showed that AtZIP1, AtZIP5, AtZIP9, AtZIP12, and AtIRT3 were induced by zinc deficiency, so it was speculated that these genes might enhance zinc uptake under zinc deficiency (Kramer U, Talke IN, Hanikenne M. Transition metal transport. FEBS Lett 2007, 581(12):2263-2272.).

Corn (Zea mays) is an important grain, feed and economic crop in China. Increasing the content of trace elements such as zinc and iron in corn grains is particularly important for improving diet or feed utilization efficiency, promoting economic development and human health. At present, it is known that many transporters are involved in the balance network system of zinc and iron ions in plants, among which the ZIP (Zinc-regulated transporters, Iron-regulated transporter-like proteins, ZIP) gene family absorbs and transports divalent metal ions such as zinc and iron. and storage play an important role. In Arabidopsis, rice, barley, and soybean, some studies on ZIP family genes have been reported, but the specific mechanism of action of ZIP family genes in plants is not yet fully understood. There are few reports on ZIP family genes. Understanding the absorption, transportation, distribution and regulation mechanism of Zn ²⁺ and Fe ²⁺ in maize will help improve the growth and development of maize in zinc and iron-deficient environments, and provide a basis for further revealing the ZIP family genes in maize. The mechanism of action lays the foundation, provides candidate genes for high-efficiency transgenic breeding of zinc and iron in corn, and also provides a good foundation for human zinc and iron nutrition.

Contents of the invention

One of the objects of the present invention is to provide the zinc-iron-regulated transporter gene isolated from corn (Zea mays);

The second object of the present invention is to provide the protein encoded by the zinc-iron regulated transporter gene;

The third object of the present invention is to apply the zinc-iron-regulated transporter gene to regulate the absorption, transport or storage of metal ions such as zinc-iron by plants, to relieve the poisoning of excessive zinc-iron to plants or to increase the amount of zinc in grain crops. iron content.

Above-mentioned purpose of the present invention is achieved through the following technical solutions:

The zinc-iron regulator transporter ZmIRT1 gene isolated from corn (Zea mays), its cDNA sequence is shown in (a), (b) or (c):

(a), the nucleotide shown in SEQ ID No.1;

(b), the nucleotide encoding the amino acid shown in SEQ ID No.2;

(c) A nucleotide capable of hybridizing to the complementary sequence of SEQ ID NO: 1 under stringent hybridization conditions, and the protein encoded by the nucleotide has the function of a zinc-iron-regulated transporter.

The "stringent hybridization conditions" mean conditions of low ionic strength and high temperature known in the art. Typically, under stringent conditions, a probe hybridizes to its target sequence to a detectably higher degree (eg, at least 2-fold over background) than to other sequences. Stringent hybridization conditions are sequence-dependent, and in different circumstances The conditions will be different, and longer sequences hybridize specifically at higher temperatures. The target sequence that is 100% complementary to the probe can be identified by controlling the stringency of hybridization or washing conditions. For detailed guidance on nucleic acid hybridization, refer to relevant literature (Tijssen, Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Probes, "Overview of principles of hybridization and the strategy of nuclear acidassays.1993). More specifically, the stringent conditions are usually selected to be lower than the specified ionic strength for specific sequences The thermal melting point (Tm) at pH is about 5-10° C. The Tm is the temperature at which 50% of the probes complementary to the target hybridize to the target sequence in equilibrium (under specified ionic strength, pH and nucleic acid concentration) ( Because the target sequence is present in excess, 50% of the probes are occupied at equilibrium at the Tm). Stringent conditions can be those in which the salt concentration is below about 1.0 M sodium ion concentration at pH 7.0 to 8.3, typically A sodium ion concentration (or other salt) of about 0.01 to 1.0 M, and a temperature of at least about 30° C. for short probes (including but not limited to, 10 to 50 nucleotides) and at least about 30° C. for long probes (including (but not limited to) greater than 50 nucleotides) is at least about 60°C. Stringent conditions can also be achieved by adding destabilizing agents such as formamide. For selective or specific hybridization, a positive signal can be At least two-fold background hybridization, optionally 10-fold background hybridization. Exemplary stringent hybridization conditions may be as follows: 50% formamide, 5×SSC and 1% SDS, incubated at 42°C; or 5×SSC, 1% SDS , incubated at 65° C., washed in 0.2×SSC and washed in 0.1% SDS at 65° C. The washing can be performed for 5, 15, 30, 60, 120 min or longer.

Preferably, the cDNA sequence of the ZmIRT1 gene isolated from maize in the present invention is shown in SEQ ID No.1.

The second object of the present invention is to provide a protein capable of absorbing, transporting or storing zinc and iron encoded by the above-mentioned zinc-iron regulator transporter ZmIRT1 gene, the amino acid sequence of the protein is shown in (a) or (b):

(a), the amino acid shown in SEQ ID No.2;

(b) A protein variant that still has the function of a zinc-iron-regulated transporter derived from the amino acid shown in SEQ ID No.2 through the substitution, deletion or/and insertion of one or more amino acid residues;

Particularly preferably, the amino acid sequence of the zinc-iron regulated transporter of the present invention is shown in SEQ ID No.2.

The "multiple" usually means 2-8, preferably 2-4, depending on the position of the amino acid residue or the type of amino acid in the three-dimensional structure of the zinc-iron regulator transporter; the "replacement" is Refers to the replacement of one or more amino acid residues with different amino acid residues; the "deletion" refers to the reduction of the number of amino acid residues, that is, the lack of one or more amino acid residues; "Insertion" refers to an alteration in the sequence of amino acid residues that results in the addition of one or more amino acid residues relative to the native molecule.

The protein variants described in the present invention can be produced by genetic polymorphism or human manipulation, and these manipulation methods are generally understood in the art. For example, amino acid sequence variants or fragments of zinc-iron regulated transporters can be prepared by mutation of DNA, wherein methods for mutagenesis or modification of polynucleotides are known in the art. Among them, a conservative substitution is to replace one amino acid residue with another amino acid with similar properties. Therefore, the zinc-iron regulated transporter and its coding gene in the present invention include both naturally occurring sequences and variants. "Variants" means substantially similar sequences, and with respect to polynucleotides, variants comprise deletions, insertions, and/or substitutions of one or more nucleotides at one or more sites in the native polynucleotide. With respect to polynucleotides, conservative variants include those that do not alter the encoded amino acid sequence due to the degeneracy of the genetic code. Naturally occurring variants such as these can be identified by available molecular biology techniques. Variant polynucleotides also include polynucleotides of synthetic origin, such as polynucleotide variants obtained by site-directed mutagenesis that still encode amino acids shown in SEQ ID No. 2, or by recombinant methods (such as DNA recombinant rows, etc.). Those skilled in the art can screen or evaluate the function or activity of the protein encoded by the variant polynucleotide through the following molecular biological techniques: DNA binding activity, interaction between proteins, activation of gene expression in transient studies or transgenic plants The effect expressed in etc.

The present invention isolates and clones the ZmIRT1 gene of the zinc-iron regulator transporter from corn, and the experimental results of subcellular localization show that the protein encoded by the ZmIRT1 gene is located in the plasma membrane and endoplasmic reticulum, so it is speculated that it is related to the detoxification and storage of zinc and iron ions , in plant cells to absorb, transport and store zinc and iron, and participate in the detoxification of zinc and iron ions. On the basis of subcellular localization, the present invention further analyzed the expression of corn embryos and endosperms at different days after pollination, and found that the expression of ZmIRT1 was up-regulated in iron-deficient roots and shoots, and the expression level of ZmIRT1 in shoots of zinc-deficient 96h increase, indicating that the ZmIRT1 gene has the function of regulating the absorption and transport of zinc and iron in plants (including underground parts and aerial parts); in addition, the ZmIRT1 gene is mainly expressed in the embryo, and the expression of ZmIRT1 is up-regulated in the later stage of embryo development, indicating that ZmIRT1 is related to the embryo It is related to the ripening and participates in the regulation of the accumulation of zinc or iron content in the grain.

Yeast complementation experiments show that the ZmIRT1 gene isolated by the present invention is similar to the ZIP gene in rice and Arabidopsis, and has the function of zinc and iron transport.

At present, it is known that many transport proteins are involved in the zinc and iron ion balance network system in plants, and some proteins have also been applied to plant transgenic research. For example, overexpressing the AtZIP1 gene in barley can increase the content of zinc and iron in seeds (Ramesh SA, Choimes S, Schachtman DP. Over-expression of an Arabidopsis zinc transporter in hordeum vulgare increases short-term zinc uptake after zinc deprivation and seed zinc content. Plant molecular biology 2004,54(3):373-385.), also, over Over-expression of OsIRT1 leads to increased iron and zincaccumulations in rice. Plant, cell & environment 2009, 32 (4):408-416.). However, overexpression of OsZIP4, OsZIP5, OsZIP8, and OsZIP9 in rice resulted in excess Zn accumulation in roots and reduced Zn content in shoots of plants (Lee S, Kim SA, Lee J, et al. Zinc deficiency-inducible OsZIP8encodesa plasma membrane-localized zinc transporter in rice.Molecules and cells2010,29(6):551-558; Lee S, Jeong HJ, Kim SA, et al.OsZIP5is a plasma membranezinc transporter in rice.Plant molecular biology 2010,73(4- 5):507-517; Ishimaru Y, Masuda H, Suzuki M, et al. Overexpression of the OsZIP4zinctransporter confers disarrangement of zinc distribution in riceplants. Journal of experimental botany 2007,58(11):2909-2915.) did not reach The purpose of increasing Zn content in grain, therefore, the overexpression of these genes is unfavorable for Zn enrichment in rice grain. These results suggest that ectopic overexpression may play a role in the accumulation and distribution of Zn and Fe. However, there are few studies on the zinc-iron transporter in grain.

The present invention understands that ZmIRT1 is mainly expressed in the embryo through the expression pattern of the ZmIRT1 gene in the embryo and endosperm, so overexpressing the ZmIRT1 gene of the present invention in grain crop seeds can increase the accumulation of zinc and iron content in the grain.

Therefore, the present invention provides a method for regulating the ability of plants to absorb, transport or store zinc and iron, to remove excessive zinc and iron to plants, to regulate the development or maturation of embryos or endosperms, and to increase the content of zinc and iron in grain crops, including : the ZmIRT1 gene of the present invention is operably connected with the expression control element to obtain a recombinant plant expression vector; the recombinant plant expression vector is transformed into the plant, and the ZmIRT1 gene is overexpressed in the plant; particularly, the ZmIRT1 gene of the present invention is used in the grain When it is overexpressed in crop seeds, it can effectively increase the content of zinc and iron in grain crop seeds.

The present invention further provides a recombinant plant expression vector containing the zinc-iron regulated transporter gene and a host cell containing the recombinant plant expression vector.

The zinc-iron-regulated transporter gene is operably connected with the expression control element to obtain a recombinant plant expression vector capable of expressing the zinc-iron-regulated transporter gene in plants. "Operably linked"refers to a functional linkage between two or more elements, which may be contiguous or non-contiguous. For example, the recombinant plant expression vector may consist of a 5' non-coding region, a nucleotide shown in SEQ ID No.1 and a 3' non-coding region, wherein the 5' non-coding region may include a promoter sequence, enhancer sequence or/and translation enhancing sequence; the promoter can be a constitutive promoter, an inducible promoter, a tissue or organ-specific promoter; the 3' non-coding region can contain a terminator sequence , mRNA cleavage sequence, etc. A suitable terminator sequence can be taken from the Ti-plasmid of Agrobacterium tumefaciens, such as the octopine synthase or nopaline synthase termination region. For example, in order to specifically express the zinc-iron-regulated transporter gene of the present invention in food crop seeds, the zinc-iron-regulated transporter gene can be connected under the seed-specific expression promoter to construct a recombinant plant expression vector, and the recombinant After the plant expression vector transforms the recipient plant, the zinc-iron-regulated transporter gene can be specifically expressed in the seed of the recipient plant, so as to achieve the effect of increasing the zinc-iron content of the seed or regulating embryo development.

In addition, those skilled in the art can optimize the nucleotide sequence shown in SEQ ID No. 1 to enhance its expression in plants. For example, polynucleotides can be synthesized by optimizing the preferred codons of the target plant to enhance the expression level of the gene in the target plant, and these methods are well known to those skilled in the art.

In addition, the recombinant plant expression vector may also contain a selectable marker gene for selection of transformed cells. Selectable marker genes are used to select transformed cells or tissues. The selectable marker genes include: genes encoding antibiotic resistance and genes conferring resistance to herbicide compounds, etc. In addition, the marker genes also include phenotypic markers, such as β-galactosidase and fluorescent protein.

The "transformation" refers to genetically transforming polynucleotides or polypeptides into plants by introducing genes into plant cells. Methods for introducing the polynucleotide or polypeptide into plants are well known in the art, including but not limited to stable transformation methods, transient transformation methods, and virus-mediated methods. "Stable transformation" means that the introduced polynucleotide construct is integrated into the genome of the plant cell and can be inherited by its progeny; "transient transformation" means that the polynucleotide construct is introduced into the plant but only temporarily expressed in the plant or exist.

Transformation protocols and protocols for introducing the polynucleotide into plants may vary depending on the type of plant (monocot or dicot) or plant cell used for transformation. Suitable methods for transforming plant cells with the polynucleotide include: microinjection, electroporation, Agrobacterium-mediated transformation, direct gene transfer, and high-speed ballistic bombardment, among others. In specific embodiments, the ZmIRT1 gene of the invention can be provided to plants using a variety of transient transformation methods. In other embodiments, the ZmIRT1 gene of the invention can be introduced into a plant by contacting the plant with a virus or viral nucleic acid. Typically, such methods involve introducing a ZmIRT1 gene construct of the invention into a viral DNA or RNA molecule.

Transformed cells can be regenerated into stably transformed plants using conventional methods (McCormick et al. Plant Cell Reports. 1986.5:81-84). The present invention can be used to transform any plant species, including but not limited to: monocotyledonous plants or dicotyledonous plants; preferably, the target plants include food crops, vegetables or fruit trees, etc., more preferably food crops, for example, can be corn , rice, barley, wheat, sorghum, soybeans, potatoes and other food crops.

Definition of terms involved in the present invention

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods, devices and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods, devices and materials are now described.

The term "recombinant host cell strain" or "host cell" means a cell comprising a polynucleotide of the invention, regardless of the method used for insertion to produce a recombinant host cell, such as direct uptake, transduction, pairing, or in the art. other known methods. Exogenous polynucleotides may remain as non-integrating vectors such as plasmids or may integrate into the host genome. The host cell can be a prokaryotic cell or a eukaryotic cell, and the host cell can also be a monocotyledonous or dicotyledonous plant cell.

The term "nucleotide" means deoxyribonucleotides, deoxyribonucleosides, ribonucleosides or ribonucleotides and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids that contain known analogs of natural nucleotides that have binding properties similar to the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless specifically limited otherwise, the term also means oligonucleotide analogs, including PNA (peptide nucleic acid), DNA analogs used in antisense technology (phosphorothioate, phosphoramidate, etc.) . Unless otherwise specified, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (including, but not limited to, degenerate codon substitutions) and complementary sequences as well as the explicitly designated sequences. In particular, degenerate codon substitutions can be achieved by generating sequences in which one or more selected (or all) codons are substituted at position 3 with mixed bases and/or deoxyinosine residues (Batzer et al. , Nucleic Acid Res.19:5081 (1991); Ohtsuka et al., J.Biol.Chem.260:2605-2608 (1985); and Cassol et al., (1992); Rossolini et al., Mol Cell.Probes 8: 91-98 (1994)).

The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to mean a polymer of amino acid residues. That is, descriptions for polypeptides apply equally to descriptions of peptides and descriptions of proteins, and vice versa. The term applies to naturally occurring amino acid polymers as well as amino acid polymers in which one or more amino acid residues is a non-naturally encoded amino acid. As used herein, the term encompasses amino acid chains of any length, including full-length proteins (ie, antigens), wherein the amino acid residues are linked via covalent peptide bonds.

Description of drawings

Figure 1 Schematic diagram of yeast expression vector pFL61.

Fig. 2 The expression pattern of ZmIRT1 under standard Hoagland medium conditions; S (shoot), R (root).

Fig. 3 Expression pattern of ZmIRT1 gene under various treatment conditions.

Fig. 4 The expression pattern of ZmIRT1 gene during the development of maize embryo and endosperm.

Fig. 5 Phylogenetic tree analysis of ZIP members in different species.

Fig. 6 Restriction restriction identification of pRTL2NGFP-ZmIRT1 recombinant vector.

Fig. 7 Flow chart of pRTL2NGFP-ZmIRT1 vector construction.

Fig. 8 The subcellular localization of ZmIRT1 in onion epidermal cells; GFP is the localization of pRTL2NGFP empty vector; ZmIRT1 is the subcellular localization of pRTL2NGFP-ZmIRT1.

Figure 9 The subcellular localization of ZmIRT1 in Arabidopsis mesophyll protoplasts; GFP is the localization of pRTL2NGFP empty vector; ZmIRT1 is the subcellular localization of pRTL2NGFP-ZmIRT1.

Figure 10 pFL61-ZmIRT1 and pFL61-OsZIP5, pFL61-OsZIP8, pFL61-OsZIP8, pFL61-OsIRT1 positive connection recombinant vector enzyme digestion identification; M is a 1Kb Marker; 1-4 are pFL61-ZmIRT1, pFL61-OsZIP5, pFL61-OsZIP8, pFL61 - OsIRT1 double enzyme digestion results.

Fig. 11 Flow chart of pFL61-ZmIRT1 vector construction.

Figure 12 ZmIRT1 yeast complementation experiment results.

Fig. 13 Schematic diagram of plant expression vector pBI121-ZmIRT1.

Figure 14 ZmIRT1 gene overexpression in Arabidopsis increases the experimental results of zinc or iron content in Arabidopsis; ZmIRT1 is the result of the determination of iron and zinc content in Arabidopsis seeds transfected with ZmIRT1 gene; WT is the iron and zinc content in wild-type Colombian seeds The results of the assay.

detailed description

The present invention will be further described below in conjunction with specific embodiments, and the advantages and characteristics of the present invention will become clearer along with the description. However, these embodiments are only exemplary and do not constitute any limitation to the scope of the present invention. Those skilled in the art should understand that the details and forms of the technical solutions of the present invention can be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the protection scope of the present invention.

Experimental Materials

1.1 Plant material

The maize inbred line X178 was provided by Hebei Agricultural College/National Corn Improvement Center Hebei Branch Laboratory of Hebei Agricultural University, and the rice inbred line Nipponbare was donated by the School of Life Sciences, Beijing Normal University.

1.2 Strains and vectors

Escherichia coli (E.coli) strain Mach1-T1 and Agrobacterium (A.tumefacterium) strains EHA105 and GV3101 were preserved by our laboratory. The pGEM-Teasy vector was purchased from Promega. Yeast expression vector pFL61 (see Figure 1 for a schematic diagram), yeast strain zrt1zrt2ZHY3 (MATαade6can1his3leu2trp1ura3zrt1::LEU2zrt2::HIS3), fet3fet4DEY1453 (MATa/MATa ade2/+can1/can1his3/his3leu2/leu2trp1/trp1ura3/ura3fet3-2::3HIS3/ -2::HIS3fet4-1::LEU2/fet4-1::LEU2), DY1455 (MATaade6can1his3leu2trp1ura3) were kindly donated by Professor Zhang Hongsheng of Nanjing Agricultural University.

Example 1 Cloning of ZmIRT1 gene of maize zinc and iron regulated transporter

1. Processing of plant material

First soak the vermiculite with Hoagland nutrient solution, sow the seeds of the corn inbred line X178 on the seedling tray, cover the top with a layer of dry vermiculite, and cultivate them in a greenhouse (16h light/8h dark, 26°C). When it grows to 2 leaves and one heart, it is moved into the standard Hoagland nutrient solution and grows for 6 days to 3 leaves and one heart (the nutrient solution is changed every 3 days). After treatment under high zinc and iron conditions for 0, 6, 12, 24, 48, and 96 hours, the shoots and roots of the seedlings were harvested, respectively, and stored at -80°C for total RNA extraction after quick freezing in liquid nitrogen.

2. Extraction of corn total RNA

The total RNA of maize was extracted by Trizol method.

3. Synthesis of cDNA

(1) To remove DNA, prepare the reaction system as follows: total RNA (1μg/μL) 1.0μL, DNAse I (10U/μL) 1.0μL, 10×DNAse I buffer 1.0μL, DEPC H ₂ O 7.0μL, total 10.0 μL; 37°C for 30 minutes, add 1 μL of 25mM EDTA, and stop the reaction at 65°C for 5 minutes.

(2), add 1μL oligo(dT18), 65℃ for 5min;

(3) 12 μL of the above, and then add the following components to obtain a reverse transcription system: 5× reaction buffer 4.0 μL, Ri RT (20U/μL) 1.0 μL, Re RT (200U/μL) 1.0 μL, 10mM dNTP mix 2.0 μL, total: 20.0 μL; 60 min at 42°C, 5 min at 70°C, stop the reaction.

4. Cloning of the target gene

(1), design primers according to the ORF frame of the target gene:

ZmIRT1F 5'- GCGGCCGCTCTAGA ATGTCTTGGCGGCGAAACC-3'NotI,XbaI

ZmIRT1R 5'- GCGGCCGCTACGTA TCA CGCCCACTTGGCCATGATG-3'NotI,SnaBI

Using the cDNA in the above step 3 as a template, select ExTaq enzyme and 2×GCI buffer for PCR amplification. The PCR program is: 95°C pre-denaturation for 4 minutes; 94°C denaturation for 1 minute, 60°C annealing for 1 minute, 72°C extension for 1 minute, 33 cycles ; Extend at 72°C for 10 minutes;

(2), clone the obtained fragment into the pGEM-T vector, and transform the Mach1-T1 strain;

(3) The positive recombinant plasmid was identified by enzyme digestion, named pGEM-ZmIRT1, and correctly cloned by sequencing. The cloned gene was named ZmIRT1, the cDNA sequence of the gene is shown in SEQ ID No.1, the deduced amino acid sequence is shown in SEQ ID No.2, and its whole genome sequence is shown in SEQ ID No.3.

Example 2 Expression patterns of ZmIRT1 in seedlings, embryos and endosperms

The reverse transcribed cDNA in Step 3 of Example 1 was diluted 10 times as a template for the PCR reaction, and the PCR reaction system was as follows:

cDNA 2.0 μL, ExTaq 0.1 μL, 2×GCI buffer 10.0 μL, 10 mM dNTP mix 0.8 μL, upstream primer RTZmIRT1F (10 μM/μL) 1.0 μL, downstream primer RTZmIRT1R (10 μM/μL) 1.0 μL, ddH ₂ O 5.1 μL, 20.0 μL in total;

RTZmIRT1F 5'-CACCACCTTCGTCGCCAT-3'

RTZmIRT1R 5'-TGTTGCCACCCTTCCTCC-3'

PCR reaction conditions: pre-denaturation at 94°C for 4 minutes; 30 cycles, each cycle of denaturation at 94°C for 45 seconds, annealing at 60°C for 1 minute, extension at 72°C for 1 minute; finally extending at 72°C for 10 minutes, cooling to 16°C, taking out the PCR product and putting it in Store at 4°C.

Detection of the expression level of the target gene: the reverse-transcribed cDNA in step 3 of Example 1 was diluted 20 times as a template for the Real-timePCR reaction, Actin was used as an internal reference, and the reaction system was as follows:

cDNA 5.0 μL, SYBR Green I 10.0 μL, Rox 0.4 μL, upstream primer ZmActin1F (10 μM/μL) 0.4 μL, downstream primer ZmActin1R (10 μM/μL) 0.4 μL, ddH ₂ O 3.8 μL, total 10.0 μL;

ZmActin1F 5'-ATGTTTCCTGGGATTGCCGAT-3'

ZmActin1R 5'-CCAGTTTCGTCATACTCTCCCTTG-3'

The program used: 95°C for 2min, 95°C for 15sec, 60°C for 34sec, 40 cycles, and the expression level was calculated by the ΔΔCt method.

Through real-time RT-PCR expression analysis, it was found that under normal nutritional conditions, ZmIRT1 was expressed in both aboveground and underground parts, and the expression level was higher in roots (Figure 2). Under zinc deficiency conditions, ZmIRT1 was expressed in 96h aboveground Up-regulation; the expression level of ZmIRT1 in the above-ground part gradually increased under high zinc conditions, and reached the highest level at 96h, however, the expression level in the underground part decreased (Figure 3). These results indicated that ZmIRT1 was sensitive to Zn concentration during the seedling stage.

Under iron-deficiency conditions, the expression of ZmIRT1 was up-regulated at 96 h in the above-ground parts, and the expression in the underground parts was significantly increased. Concentration is more sensitive.

The expression analysis of embryos and endosperms at different days after pollination found that the expression level of ZmIRT1 was significantly up-regulated mainly in the later stage of embryo development (Figure 4), indicating that ZmIRT1 played a certain role in the development of embryos.

Example 3 Bioinformatics analysis of ZmIRT1

Bioinformatics analysis showed that ZmIRT1, a zinc-iron-regulated transporter, was localized on the first chromosome of maize. ZmIRT1, a zinc-iron-regulated transporter, consisted of 381 amino acids and contained 8 transmembrane domains. There is a histidine-rich variable region between them, which may be related to the binding and transport of metal ions. Phylogenetic tree analysis showed that ZmIRT1 had a close evolutionary relationship with AtIRT1, AtIRT2, OsIRT1, and OsIRT2 (Fig. 5), suggesting that ZmIRT1 might be an iron transporter.

Example 4 Subcellular localization of ZmIRT1

1. Construction of fusion expression vector

Primers were designed according to the sequence of the ZmIRT1 gene, and the primer sequences were as follows:

ZmIRT1GF 5'- CTCGAG ATGTCTTGGCGGCGAAACC-3'XhoI

ZmIRT1GR 5'- TCTAGA CGCCCACTTGGCCATGATG-3'XbaI

Add a suitable restriction site, and remove the stop codon at the 3' end of the gene, use the plasmid that was connected to the pGEM-T vector and sequenced correctly when cloning the gene as a template, select ExTaq enzyme and 2×GCI buffer for PCR amplification, PCR The program was: pre-denaturation at 95°C for 4 min; denaturation at 94°C for 1 min, annealing at 60°C for 1 min, extension at 72°C for 1 min, 33 cycles; extension at 72°C for 10 min. The amplified fragment was recovered by 1% agarose gel electrophoresis and cloned into pGEM-T vector, transformed into Escherichia coli strain Mach1-T1, and positive clones were obtained by LB medium (IPTG, X-gal, Amp). Excision and sequencing verification; the sequenced correct plasmid connected to the pGEM-T carrier when cloning the gene was used as a template, and ExTaq enzyme and 2×GCI buffer were used for PCR amplification, and the amplified fragment was recovered by 1% agarose gel electrophoresis and cloned After sequencing the correct plasmid into the pGEM-T vector, construct the target fragment into the pRTL2NGFP vector and name it pRTL2NGFP-ZmIRT1. Figure 6 is the enzyme digestion identification diagram, and Figure 7 is the construction flow chart.

2. Cut the pRTL2NGFP vector with the corresponding restriction enzymes and the genes after different restriction enzymes, connect them with T ₄ DNA ligase, transform the Mach1-T1 strain, extract the plasmids, identify and select the correct recombinant plasmids for gene gun transformation Onion skin.

3. Preparation of gene gun microprojectiles

4. Transformation of onion epidermis with gene gun

From the localization results, it can be seen that the zinc-iron-regulated transporter ZmIRT1 is localized on the inner membrane of the cell (Fig. 8). In order to further determine the plasma membrane and inner membrane system of the cells, ER markers were used to transform Arabidopsis thaliana mesophyll protoplasts. The results proved that ZmIRT1, a zinc-iron-regulated transporter, was localized in the plasma membrane and endoplasmic reticulum (Fig. 9).

Example 5 Yeast Complementation Experiment

1. Construction of yeast expression vector

Design primers by adding appropriate restriction sites according to the target gene sequence:

ZmIRT1YF 5'- GCGGCCGCTCTAGA ATGTCTTGGCGGCGAAACC-3'NotI,XbaI

ZmIRT1YR 5'- GCGGCCGCTACGTA TCACGCCCACTTGGCCATGATG 3'NotI,SnaBI

Using the correctly sequenced plasmid connected to the pGEM-T vector when cloning the gene as a template, ExTaq and 2×GCIbuffer were selected for PCR amplification. The PCR program was: 95°C pre-denaturation for 4 minutes; 94°C for 1 minute; Extend 1min at ℃, 33 cycles; extend at 72℃ for 10min. The amplified fragment was recovered by 1% agarose gel electrophoresis and cloned into pGEM-T vector, transformed into Escherichia coli strain Mach1-T1, and positive clones were obtained by LB medium (IPTG, X-gal, Amp). Cutting and sequencing verification, the sequenced correct plasmids were digested accordingly, and the target fragments were constructed on the pFL61 vector, named pFL61-ZmIRT1 and pFL61-OsZIP5, pFL61-OsZIP8 and pFL61-OsIRT1, Figure 10 is the enzyme digestion identification map, Figure 11 is a construction flow chart.

The pFL61 vector was digested with NotI and the digested ZmIRT1 fragment was ligated with T ₄ DNA ligase to transform the Mach1-T1 strain, and the plasmid was digested and identified to select the correct recombinant plasmid for transformation of Saccharomyces cerevisiae.

2. Transformation of yeast by electric shock transformation method

(1) Single colonies of zrt1zrt2ZHY3, fet3fet4DEY1453 and DY1455 were picked from the YPD plate and placed in 20 mL of YPD liquid medium, cultured on a shaker at 28°C for about 24 hours;

(2) Transfer the above 2% volume of the bacterial solution to 100mL YPD medium and continue to proliferate for about 4-5 hours. When the OD ₆₀₀ of the bacterial solution is 1.2-1.5, the competent state can be prepared;

(3) Collect the bacterial liquid into a 50mL centrifuge tube, centrifuge at 5,000rpm at 4°C for 5min, and discard the supernatant;

(4) Add an equal volume of deionized water, resuspend the bacteria on ice, centrifuge at 5,000 rpm at 4°C for 5 minutes, and discard the supernatant;

(5) Add 1/2 volume of deionized water, resuspend the bacteria on ice, centrifuge at 5,000 rpm at 4°C for 5 minutes, and discard the supernatant;

(6) Add 10 mL of 1M sorbitol solution, resuspend the bacteria on ice, centrifuge at 5,000 rpm at 4°C for 5 min, and discard the supernatant;

(7) Add 450-600 μL of sorbitol solution, gently suck with the tip of the pipette with the head removed, and resuspend the bacteria;

(8) Add about 100 μL of competent state to each 1.5mL centrifuge tube, and aliquot;

(9) Add an appropriate amount of DNA (about 10 μL, c≥200 ng/μL) to each competent tube, place it on ice for 1-2 minutes, and then suck it into a pre-cooled electric shock cup without air bubbles;

(10), electric shock transformation, immediately add about 800 μ L, 1M pre-cooled sorbitol solution, resuspend the bacteria;

(11), aspirate the thalli from the electric shock cup, and apply SD/Ura-plate;

(12) Visible plaques can grow on the SD plate after cultured at 28°C for about 6 days.

3. Identification of positive yeast clones

(1) Take 1.5 mL of yeast culture, centrifuge at 9,000 rpm for 30 seconds, discard as much supernatant as possible, and collect yeast cells;

(2) Add 600 μL Sorbitol buffer, gently pipette to fully resuspend the cells, add 80 U of Lyticase, mix thoroughly by inversion, incubate at 37°C for 30 minutes to digest the cell wall, and invert several times in the middle;

(3) Centrifuge at 13,000 rpm for 1 min, discard the supernatant as much as possible, add 250 μL of solution YP1 to resuspend the bacterial pellet, and vortex until completely suspended;

(4) Add 250 μL of YP2 solution, turn over gently to fully lyse the bacteria, and place at room temperature for 4 minutes;

(5) Add 350 μL of YP3 solution, turn it over gently, a white flocculent precipitate will appear when fully mixed, let it stand on ice for 3-5 minutes, centrifuge at 13,000 rpm for 5 minutes, and carefully absorb the supernatant.

(6) Add the supernatant obtained in the previous step into the adsorption column AC (the adsorption column is placed in the collection tube), centrifuge at 12,000rpm for 30-60 seconds, and discard the waste liquid in the collection tube;

(7) Add 500 μL protein-removing solution PD, centrifuge at 12,000 rpm for 30-60 seconds, and discard the waste solution;

(8) Add 500 μL of rinse solution WB (absolute ethanol has been added), centrifuge at 12,000 rpm for 30-60 seconds, and discard the waste liquid;

(9) Add 500 μL of washing solution WB, centrifuge at 12,000 rpm for 30-60 seconds, and discard the waste liquid;

(10), put the adsorption column AC back into the empty collection tube, centrifuge at 13,000rpm for 2min, and remove the rinse solution;

(11) Take out the adsorption column AC, put it into a clean centrifuge tube, add 50 μL of elution buffer EB (65-70°C water bath) to the middle part of the adsorption membrane, place it at room temperature for 2 minutes, and centrifuge at 13,000 rpm for 1 minute.

(12) Use the extracted 1 μL DNA as a template, and the primers at both ends of the gene as PCR amplification primers, perform PCR amplification to verify the target gene, verify the correct bacterial solution, add 25% glycerol and store at -80°C.

4. Yeast Complementation Experiment

pFL61, pFL61-ZmIRT1, pFL61-OsZIP5, pFL61-OsZIP8, and pFL61-OsIRT1 were transformed into yeast mutant strains zrt1zrt2ZHY3 and fet3fet4DEY1453, respectively, pFL61 was used as a negative control, and OsZIP5 and OsZIP8 were used as positive controls for zinc transporters (Lee S, Kim SA, Lee J, et al.Zinc deficiency-inducible OsZIP8encodes plasma membrane-localized zinc transporter in rice.Molecules and cells 2010,29(6):551-558; Lee S,Jeong HJ,Kim SA,et al.OsZIP5is a plasma membrane zinc transporter in rice.Plant molecular biology 2010,73(4-5):507-517; Ishimaru Y,Masuda H,Suzuki M,et al.Overexpression of the OsZIP4zinc transporter confers disarrangement of zinc distribution in rice plants.Journal of experimentalbotany 2007,58 (11):2909-2915.), OsIRT1 is the positive control of iron transporter (Lee S, An G.Over-expression of OsIRT1 leads to increased iron and zinc accumulations inrice.Plant, cell&environment 2009,32(4):408- 416.), pFL61 transformed wild-type strain DY1455 was used as another positive control, and the yeast identified as positive after transformation were cultured in SD liquid medium, and the yeast liquid was diluted to 4 concentrations (OD ₆₀₀ =1, 0.1, 0.01 , 0.001), and then take 5 μL points in low zinc, low iron and normal SD medium, low zinc medium (SD medium added 0.4mM EDTA, 0.4mM EDTA and 250μM ZnSO ₄ , 0.4mM EDTA and 300μM ZnSO ₄ ) , low iron medium (SD medium added 50mM MES, 50mM MES and 50μM FeCl ₃ , 50mM MES and 100μM MFeCl ₃ ) Yeast complementation refers to the experiment of Lin and YF (Lin YF, Liang HM, Yang SY, et al. Arabidopsis IRT3 is a zinc-regulated and plasma membrane localized zinc/irontransporter. The New phytologist 2009,182(2):392-404 .) method, cultivated at 28°C, and observed the test results for 6 days.

The results of yeast complementation experiments showed that DY-pFL61 (wild type), Z-ZmIRT1, Z-OsZIP5, Z-OsZIP8, and Z-OsIRT1 could be clearly observed in the medium supplemented with 250 μM ZnSO ₄ under low zinc conditions. The vector pFL61 grows better; moreover, ZmIRT1 grows better than the reported rice OsZIP. Under low iron conditions, the transport activity of D-ZmIRT1 is stronger than that of OsIRT1 that has been reported (Fig. 12). It plays a major role in the absorption and transport of zinc and iron during growth and development.

Experimental example 1 The experiment of overexpressing ZmIRT1 gene in Arabidopsis to increase the content of iron and zinc in Arabidopsis seeds

The ZmIRT1 gene was connected with the plant expression vector pBI121 controlled by the constitutive 35S promoter to construct the ZmIRT1 gene recombinant plant expression vector (Figure 13); the constructed recombinant plant expression vector was transformed into Arabidopsis thaliana, and the positive ZmIRT1 gene was identified Arabidopsis: positively transgenic Arabidopsis thaliana and wild-type Columbia were cultured under the same culture conditions, and the seeds of transgenic ZmIRT1 and wild-type Arabidopsis were harvested, and the transgenic Arabidopsis and wild-type The content of iron and zinc in the seeds; a certain amount of plant material was weighed, digested by microwave, constant volume, and the content of zinc and iron was determined by ICP-MS method. Each batch of 200mg seeds was measured, three batches were measured, and the average value of the three batches of data was taken. The measurement results are shown in Figure 14. From the results in Figure 14, it can be seen that overexpressing the ZmIRT1 gene in Arabidopsis can effectively increase the iron and zinc content in seeds, and the iron content is particularly increased.

Claims (8)

1. from Semen Maydiss (Zea mays) detached zinc-iron regulation and control transporter ZmIRT1 gene it is characterised in that its cDNA sequence Nucleotide sequence shown in SEQ ID No.1.

2. zinc-iron described in claim 1 regulates and controls the protein of transporter ZmIRT1 coded by said gene it is characterised in that described egg The aminoacid sequence of white matter is shown in SEQ ID No.2.

3. the recombinant expression carrier of transporter ZmIRT1 gene is regulated and controled containing zinc-iron described in claim 1.

4. according to the recombinant expression carrier described in claim 3 it is characterised in that：Described recombinant expression carrier is recombinant plant Expression vector.

5. described in claim 1, zinc-iron regulation and control transporter ZmIRT1 gene is improving plant absorption, transhipment or storage zinc or ferrum energy Application in power.

6. zinc-iron described in claim 1 regulates and controls transporter ZmIRT1 gene answering in increasing cereal crops seed zinc or iron content With.

7. zinc-iron described in claim 1 regulates and controls transporter ZmIRT1 gene in the zinc releasing excess or ferrum to answering in plant poisoning With.

8. according to the application described in claim 5-7 any one it is characterised in that including：Zinc-iron described in claim 1 is adjusted Transporter ZmIRT1 gene is exercisable is connected with expression regulation element for control, obtains recombinant plant expression vector；Restructuring is planted Thing expression vector transformation receptor plant or plant cell, cultivate and obtain transgenic plant.