CN104212814A

CN104212814A - Zea mays zinc iron-regulated transporter ZmZIP4 and ZmZIP6 genes and applications thereof

Info

Publication number: CN104212814A
Application number: CN201410239195.5A
Authority: CN
Inventors: 李素贞; 李宏博; 陈景堂; 黄亚群; 祝丽英
Original assignee: Hebei Agricultural University
Current assignee: Hebei Agricultural University
Priority date: 2013-05-31
Filing date: 2014-05-30
Publication date: 2014-12-17

Abstract

The invention discloses Zea mays zinc iron-regulated transporter ZmZIP4 and ZmZIP6 genes and applications thereof. The Zea mays zinc iron-regulated transporter ZmZIP4 and ZmZIP6 genes are separated from Zea mays, and the cDNA sequences of the genes are represented by SEQ ID No.1 and SEQ ID No.3 respectively. Subcellular localization shows that a zinc iron-regulated transporter encoded by the two genes is localized in the plasma membrane and endoplasmic reticulum of a cell. Quantitative real-time RT-PCR expression analysis shows that the ZmZIP4 plays a role in the embryo growth process, and the ZmZIP6 is related to embryo maturation. Yeast complementation experiments show that each of the ZmZIP4 and the ZmZIP6 has a zinc and iron transportation function. The separated ZmZIP4 and ZmZIP6 genes have important application prospects in the regulation of zinc and iron absorption, transportation and storage of plants, and promotion of the embryo maturation, and radicle and germ growth.

Description

Corn zinc-iron regulation and control transporter ZmZIP4 and ZmZIP6 gene and application thereof

Technical field

The present invention relates to the metal-ions transportation body gene be separated from plant, particularly relate to that be separated from corn with absorption that is zinc-iron, transhipment or store relevant regulation and control transporter ZmZIP4 and ZmZIP6 gene, the invention further relates to regulation and control transporter ZmZIP4 or ZmZIP6 gene absorbs at regulating plant, transhipment or store zinc or iron ability, promote the growth of embryo or application that is ripe and that increase in food crop seed zinc iron content, belong to separation and the Application Areas of plant metal ion regulation and control transporter gene.

Background technology

Zinc and iron are the necessary trace elements of organism, important role (Wintz H in the growth and development process of plant, Fox T, Wu YY, et al.Expression profiles of Arabidopsis thaliana in mineral deficiencies reveal novel transporters involved in metal homeostasis.The Journal of biological chemistry2003,278 (48): 47644-47653.).Zinc is structure cofactor (the Haydon MJ of organism more than 300 kind of enzyme and key protein, Cobbett CS.A novel major facilitator superfamily protein at the tonoplast influences zinc tolerance and accumulation in Arabidopsis.Plant physiology2007,143 (4): 1705-1719.).Zinc not only participates in the various metabolism of body, microbial film stablize with the physiological functions such as gene expression regulation in be also responsible for important role (Mathews WR, Wang F, Eide DJ, et al.Drosophila fear of intimacy encodes 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.).The shortage of zinc can cause the Oxidative demage of chlorophyll, lipid, albumen, plasma membrane, and the appropriate content increasing zinc in plant materials can improve crop yield, and in plant materials, the excessive accumulation of zine ion can produce murder by poisoning to plant.

Iron plays a significant role in the catalytic reaction process of cellular respiration, photosynthesis and metalloprotein, and be important electron transit mediator, therefore, ferro element has irreplaceable function in protokaryon and Eukaryotic vital movement.In addition, too high in cell Fe ³⁺/ Fe ²⁺oxidation-reduction potential can cause the generation of super oxygen compound, to cell damage (Briat JF, Lebrun M.Plant responses to metal toxicity.Comptes rendus de l'Academie des sciences Serie III, Sciences de la vie1999,322 (1): 43-54.).Therefore, the strict balance controlling metal ion in plant materials is vital.

The albumen participating in zinc-iron absorption mainly contains three classes, all exist with protein family form, comprising: ZIP, be i.e. zinc regulation and control transporter (Zinc-regulated transporter, ZRT) and iron regulation and control transporter (Iron-regulated transporter, IRT).Yeast function complementation experiment display ZIP family gene can be transported and comprise Zn ²⁺, Fe ²⁺, Cu ²⁺, Cd ²⁺at interior many kinds of metal ions (Colangelo EP, Guerinot ML.Put the metal to the petal:metal uptake and transport throughout plants.Current opinion in plant biology2006,9 (3): 322-330.).ZIP is generally made up of 309-476 amino-acid residue, there are 8 potential membrane spaning domains and similar topological framework, a long variable region is had between 3rd and the 4th cross-film district, variable region is positioned at born of the same parents, its C, N-terminal are positioned at outside born of the same parents, histidine residues is rich in this district, may with the combination of metal, transport relevant (Guerinot ML.The ZIP family of metal transporters.Biochim Biophys Acta2000,1465 (1-2): 190-198.).

In the plants such as Arabidopis thaliana, paddy rice, puncture vine, clover, soybean, wild-type emmer wheat, grape, identify ZIP gene and its function is studied at present.16 ZIP family genes are found in Arabidopis thaliana, AtIRT1 is separated by yeast complementation experiment first the ZIP functional gene obtained, it is mainly expressed at root, and the process LAN of this gene can cause excessive accumulation (the Eide D of nickel, 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 America1996, 93 (11): 5624-5628, Henriques R, Jasik J, Klein M, et al.Knock-out of Arabidopsis metal transporter gene IRT1results in iron deficiency accompanied by cell differentiation defects.Plant molecular biology2002,50 (4-5): 587-597, Varotto C, Maiwald D, Pesaresi P, et al.The metal ion transporter IRT1is necessary for iron homeostasis and efficient photosynthesis in Arabidopsis thaliana.The Plant journal:for cell and molecular biology2002,31 (5): 589-599, Vert G, Grotz N, Dedaldechamp F, et al.IRT1, an Arabidopsis transporter essential for iron uptake from the soil and for plant growth.Plant Cell2002,14 (6): 1223-1233, Nishida S, Tsuzuki C, Kato A, et al.AtIRT1, the primary iron uptake transporter in the root, mediates excess nickel accumulation in Arabidopsis thaliana.Plant & cell physiology2011,52 (8): 1433-1442.).AtIRT2 mainly expresses at root, be positioned at vesica, infer function of detoxification (the Vert G with excess metal element in cell, Briat JF, Curie C.Arabidopsis IRT2gene encodes a root-periphery iron transporter.The Plant journal:for cell and molecular biology2001,26 (2): 181-189; Vert G, Barberon M, Zelazny E, et al.Arabidopsis IRT2cooperates with the high-affinity iron uptake system to maintain iron homeostasis in root epidermal cells.Planta2009,229 (6): 1171-1179.14,15).AtIRT3 energy complementary zinc, iron transfer double-mutant, process LAN AtIRT3 can make zinc, and portion and iron accumulate (Lin YF at underground part on the ground, Liang HM, Yang SY, et al.Arabidopsis IRT3is a zinc-regulated and plasma membrane localized zinc/iron transporter.The New phytologist2009,182 (2): 392-404.).Expression analysis shows, AtZIP1, AtZIP5, AtZIP9, AtZIP12 and AtIRT3 induce by lacking zinc, infer thus, these genes may strengthen receptivity (the Kramer U of zinc under scarce zink rod part, Talke IN, Hanikenne M.Transition metal transport.FEBS Lett2007,581 (12): 2263-2272.).

Corn (Zea mays) is the important grain of China, feed and cash crop, increases the content of the trace element such as zinc, iron in corn kernel, to raising diet or food utilization efficiency, promote the development of economy and HUMAN HEALTH particularly important.At present, known many translocators take part in zinc-iron ionic equilibrium network system in plant materials, wherein ZIP (Zinc-regulated transporters, Iron-regulated transporter-like proteins, ZIP) gene family is to zinc, the absorption of the divalent-metal ions such as iron, transport and storage play an important role, at Arabidopis thaliana, paddy rice, barley, on soybean, some researchs about ZIP family gene are reported, but the mechanism of action concrete in plant materials to ZIP family gene is understood not yet completely, and it is less about the research report of the ZIP family gene of corn.Understand Zn ²⁺, Fe ²⁺absorption in corn, mode of transport, the regularity of distribution and regulation mechanism, corn growing in zinc, sideropenic environment is improved by contributing to, for the mechanism of action disclosing ZIP family gene in corn further lays the foundation, for corn zinc, the breeding of iron high-efficient transgenic provide candidate gene, also for the nutrition of mankind's zinc-iron provides good basis.

Summary of the invention

An object of the present invention is to provide the zinc-iron regulation and control transporter gene be separated from corn (Zea mays);

Two of object of the present invention is to provide the protein of zinc-iron regulation and control coded by transporter gene;

Three of object of the present invention is that described zinc-iron regulation and control transporter gene is applied to regulating plant to the absorption of the metal ions such as zinc-iron, transhipment or storage, promotes that radicle and embryonic development and embryo are ripe or increase zinc iron content in food crop seed.

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

The zinc-iron regulation and control transporter ZmZIP4 gene be separated from corn (Zea mays), its cDNA sequence is (a), shown in (b) or (c):

Nucleotide shown in (a) SEQ ID No.1;

Amino acid whose Nucleotide shown in (b) coding SEQ ID No.2;

C () and the complementary sequence of SEQ ID NO:1 can carry out the Nucleotide of hybridizing at stringent hybridisation conditions, the protein coded by this Nucleotide has the function of zinc-iron regulation and control transporter.

Present invention also offers the zinc-iron regulation and control transporter ZmZIP6 gene be separated from corn (Zea mays), its cDNA sequence be (a), shown in (b) or (c):

Nucleotide shown in (a) SEQ ID No.3;

Amino acid whose Nucleotide shown in (b) coding SEQ ID No.4;

C () and the complementary sequence of SEQ ID NO:3 can carry out the Nucleotide of hybridizing at stringent hybridisation conditions, the protein coded by this Nucleotide has the function of zinc-iron regulation and control transporter.

Described " stringent hybridisation conditions " means low ionic strength known in the art and the condition of high temperature.Usually, under high stringency conditions, probe and its target sequence hybridize can detection level than with other sequence hybridization can detection level be higher (such as exceedes background at least 2 times.Stringent hybridisation conditions is sequence dependent, will be different under different envrionment conditionss, longer sequence specific hybrid at relatively high temperatures.The target sequence with probe 100% complementation can be identified by the preciseness or wash conditions that control hybridization.Detailed guidance for nucleic acid hybridization can with reference to related documents (Tijssen, Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Probes, " Overview of principles of hybridization and the strategy of nucleic acid assays.1993).More specifically, described high stringency conditions is selected as usually lower than the heat fusion joint of distinguished sequence under regulation ionic strength pH (Tm) about 5-10 DEG C.Tm be in the state of the equilibrium 50% with the probe hybridization of target complementation to temperature (specifying under ionic strength, pH and nucleic acid concentration) residing during target sequence (because of the excessive existence of target sequence, thus under Tm in the state of the equilibrium the probe of 50% be occupied).High stringency conditions can be following condition: wherein at pH7.0 to 8.3 times salt concn lower than about 1.0M Na ion concentration, be generally about 0.01 to 1.0M Na ion concentration (or other salt), and temperature is at least about 30 DEG C for short probe (including, but is not limited to 10 to 50 Nucleotide), and is at least about 60 DEG C for long probe (including, but is not limited to be greater than 50 Nucleotide).The destabilizing agent of high stringency conditions also by adding such as methane amide realizes.For selectivity or specific hybrid, positive signal can be the background hybridization of at least twice, is optionally 10 times of background hybridizations.Exemplary stringent hybridisation conditions can be as follows: 50% methane amide, 5 × SSC and 1%SDS, cultivates at 42 DEG C; Or 5 × SSC, 1%SDS, cultivate at 65 DEG C, wash in 0.2 × SSC and wash in 0.1%SDS at 65 DEG C.Described washing can carry out 5,15,30,60,120min or longer time.

Preferably, the zinc-iron that the present invention is separated from corn regulates and controls the cDNA sequence of transporter ZmZIP4 gene for shown in SEQ ID No.1; The cDNA sequence of the zinc-iron regulation and control transporter ZmZIP6 gene be separated from corn is for shown in SEQ ID No.3.

Two of the object of the invention is to provide the protein that can absorb, transport or store zinc-iron being regulated and controled transporter ZmZIP4 gene and ZmZIP6 coded by said gene by above-mentioned zinc-iron;

Two of the object of the invention is achieved through the following technical solutions:

The zinc-iron regulation and control transporter of ZmZIP4 coded by said gene, its aminoacid sequence is for shown in (a) or (b):

Amino acid shown in (a) SEQ ID No.2;

(b) by the amino acid shown in SEQ ID No.2 by the replacement of one or more amino-acid residue, disappearance or/and insert and the derivative protein variant still with zinc-iron regulation and control transporter function obtained;

The zinc-iron regulation and control transporter of ZmZIP6 coded by said gene, its aminoacid sequence is for shown in (a) or (b):

Amino acid shown in (a) SEQ ID No.4;

(b) by the amino acid shown in SEQ ID No.4 by the replacement of one or more amino-acid residue, disappearance or/and insert and the derivative protein variant still with zinc-iron regulation and control transporter function obtained;

Described " multiple " mean 2-8 usually, are preferably 2-4, and this depends on the position of amino-acid residue in zinc-iron regulation and control transporter three-dimensional structure or amino acid whose kind; Described " replacement " refers to and replaces one or more amino-acid residue with different amino-acid residues respectively; Described " disappearance " refers to the minimizing of amino-acid residue quantity, that is to say and lacks one or more amino-acid residue respectively; Described " insertion " refers to the change of amino acid residue sequence, relative natural molecule, and described change causes adding one or more amino-acid residue.

Protein variant of the present invention can be produced by genetic polymorphism or manual operation, and these working method are generally this area and understand.Such as, the zinc-iron regulation and control amino acid sequence variation of transporter or fragment are prepared in the sudden change by DNA, wherein due to mutagenesis or change polynucleotide method known by this area.Wherein, conservative replacement a kind of amino-acid residue is replaced to the another kind of amino acid with similar quality.Therefore, zinc-iron regulation and control transporter of the present invention and encoding gene thereof comprise naturally occurring sequence and variant two kinds of forms." variant " means the sequence of basic simlarity, and for polynucleotide, variant comprises the disappearance of the one or more Nucleotide of one or more site in native polynucleotide, insertion or/and replace.For polynucleotide, conservative variant comprises those variants of the aminoacid sequence not changing coding due to the degeneracy of genetic code.Naturally occurring variant like this is identified by existing Protocols in Molecular Biology.Variant polynucleotides also comprises the polynucleotide in synthesis source, such as, adopts the amino acid whose polynucleotide variant of still encoding shown in SEQ ID No.2 that site-directed mutagenesis obtains, or the method (such as DNA resets) by recombinating.Those skilled in the art screen or evaluate function or the activity of albumen coded by variant polynucleotides by following molecular biotechnology means: DNA binding activity, interaction between albumen, the effect etc. expressed in the activation situation of genetic expression in instantaneous research or transgenic plant.

From Subcellular Localization result, the protein localization of the ZmZIP4 gene that the present invention is separated or ZmZIP6 coded by said gene is on plasma membrane and intercellular membrane.For determining the concrete location of intercellular membrane further, the present invention selects ER marker and ZmZIP4, ZmZIP6 to locate the conversion carrying out Arabidopis thaliana mesophyll protoplast altogether, and result proves that ZmZIP4, ZmZIP6 are positioned in cytoplasmic membrane and endoplasmic reticulum.On the basis of Subcellular Localization, found by real-time RT-PCR expression analysis, under normal nutrition condition, the expression amount in ZmZIP4, ZmZIP6 portion is on the ground higher than underground part, under high zink rod part, the expression amount of ZmZIP4 portion and underground part on the ground increases gradually, under high ferro condition, the expression amount of ZmZIP4 portion and underground part on the ground presents the trend raised gradually along with the prolongation of time, 96h reaches the highest, illustrate that the concentration of ZmZIP4 zinc and iron is to external world more responsive, have and store zinc-iron or remove the function of excessive zinc-iron to plant poisoning; No matter ZmZIP6 is under high zinc or high ferro condition, the expression amount of overground part and underground part does not all significantly change.These results show, ZmZIP4 is more responsive in the seedling concentration of period to zinc and iron, and ZmZIP6 is insensitive to zinc-iron concentration.After pollination different number of days Fetal liver cells in expression analysis find, ZmZIP4 expression amount in the embryo of 17 days after pollination raises, and the expression amount of ZmZIP6 in the pollination embryo of latter 21 days reaches the highest, therefore ZmZIP4 plays a role in the growth course of embryo, especially has important promotion or regulating and controlling effect to the growth of radicle and plumule; ZmZIP6 is relevant with the maturation of embryo, has the function promoting or regulate and control embryo maturation.

Yeast complementation experiment shows, no matter ZmZIP4 and ZmZIP6 that be separated of the present invention is in low zinc or the transhipment zinc shown under low iron bar part in various degree or ironwork, illustrates that ZmZIP4 or ZmZIP6 all has the function of transhipment zinc-iron.

At present, known many translocators take part in zinc-iron ionic equilibrium network system in plant materials, some albumen are also applied in the transgenic research of plant, such as in barley, process LAN AtZIP1 gene can increase zinc and the content of iron in seed (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 biology2004, 54 (3): 373-385.), equally, process LAN OsIRT1 gene, the content of zinc and iron portion on the ground in paddy rice, all increase in underground part and seed (Lee S, An G.Over-expression of OsIRT1leads to increased iron and zinc accumulations in rice.Plant, cell & environment2009, 32 (4): 408-416.).But, process LAN OsZIP4, OsZIP5, OsZIP8 in paddy rice, OsZIP9 result causes excessive zinc to be gathered in root, reduce Zn content (the Lee S of above-ground plant parts, Kim SA, Lee J, et al.Zinc deficiency-inducible OsZIP8encodes a 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 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 experimental botany 2007,58 (11): 2909-2915.) object increasing Zn content in seed is not reached, therefore, the process LAN of these genes is disadvantageous to the enrichment of zinc in rice grain.These results show, dystopy process LAN may play a role for the Storage and distribution of zinc-iron.But, about the research of zinc-iron translocator in seed is also little.

Therefore, the invention provides a kind of method that regulating plant absorbs, transports or store the ability of zinc-iron, comprising: being connected with expression regulation element of ZmZIP4 or ZmZIP6 genes being operational of the present invention is obtained recombinant plant expression vector; Recombinant plant expression vector is transformed in plant, process LAN ZmZIP4 or ZmZIP6 gene in plant materials, can Effective Regulation or improve the ability of target plant to the absorption of zinc-iron, transhipment or storage.

The invention provides a kind of method removed excessive zinc-iron and poison plant materials, the method comprises: being connected with expression regulation element of ZmZIP4 or ZmZIP6 genes being operational of the present invention is obtained recombinant plant expression vector; Recombinant plant expression vector is transformed in plant, process LAN ZmZIP4 or ZmZIP6 gene in plant materials.

Present invention also offers a kind of regulation and control or promote the method that plant seed radicle and plumule grow, the method comprises: being connected with expression regulation element of ZmZIP4 or ZmZIP6 genes being operational of the present invention is obtained recombinant plant expression vector; Recombinant plant expression vector is transformed in plant, process LAN ZmZIP4 or ZmZIP6 gene in plant materials; Wherein, in described expression regulation element, promotor is preferably the promotor of seed-specific expression.

Invention further provides a kind of method of regulation and control or promotion seed embryo maturation, the method comprises: being connected with expression regulation element of ZmZIP4 or ZmZIP6 genes being operational of the present invention is obtained recombinant plant expression vector; Recombinant plant expression vector is transformed in plant, process LAN ZmZIP4 or ZmZIP6 gene in plant materials; Wherein, the promotor in described expression regulation element is preferably the promotor of seed-specific expression.

Invention further provides and regulate and control the recombinant plant expression vector of transporter ZmZIP4 or ZmZIP6 gene and the host cell containing this recombinant plant expression vector containing described zinc-iron.

By being connected with expression regulation element of described zinc-iron regulation and control transporter ZmZIP4 or ZmZIP6 genes being operational, obtain the recombinant plant expression vector can expressing this zinc-iron regulation and control transporter gene in plant." exercisable connection " refers to functional connection between two or more elements, and the element of exercisable connection can be adjacent or non-adjacent.Such as, this recombinant plant expression vector can by 5 ' end non-coding region, SEQ ID No.1 or the Nucleotide shown in SEQ ID No.3 and 3 ' non-coding region composition, wherein, 5 ' described end non-coding region can comprise promoter sequence, enhancer sequence or/and translation enhancement sequences; Described promotor can be composition promotor, inducible promoter, tissue or organ specific promoters; 3 ' described non-coding region can comprise terminator sequence, mRNA cuts sequence etc.Suitable terminator sequence can take from the Ti-plasmid of agrobacterium tumefaciens, such as octopine synthase or nopaline synthase termination district.Such as, regulating and controlling transporter gene to make zinc-iron of the present invention carries out specific expressed at food crop seed, zinc-iron can be regulated and controled transporter gene be connected to seed-specific expression promotor below build obtain recombinant plant expression vector, after this recombinant plant expression vector transformation receptor plant, zinc-iron regulation and control transporter gene can carry out specific expressed in the seed of recipient plant, reaches the effect promoting that radicle and embryonic development, promotion embryo are ripe or increase seed zinc iron content or regulation and control embryonic development.

In addition, the nucleotide sequence shown in SEQ ID No.1 or SEQ ID No.3 can be optimized to strengthen its expression in plant by those skilled in the art.Such as.The preference codon of target plant can be adopted to be optimized synthetic polyribonucleotides to strengthen the expression level of this gene in target plant, and these methods are known by those skilled in the art.

In addition, this recombinant plant expression vector also can containing the selected marker for selecting transformant.Selected marker is for selecting the cell or tissue through transforming.Described selected marker comprises: the gene of encode antibiotic resistance and the gene etc. of imparting herbicidal compound resistance.In addition, described marker gene also comprises phenotypic markers, such as beta-galactosidase enzymes and fluorescin etc.

Described " conversion " to refer to polynucleotide or polypeptide genetic transformation channel genes to the inner such mode of vegetable cell in plant.The method in plant of described polynucleotide or polypeptide being incorporated into, known by this area, includes but not limited to stable conversion method, transient transformation methods and virus-mediated methods etc." stable conversion " refers to that the polynucleotide constructs be introduced into be integrated in the genome of vegetable cell and by its progeny inherit; " instantaneous conversion " refers to that polynucleotide to be introduced in plant but can only transient expression or existence in plant.

Transformation Protocol and the type of visual for the scheme of described polynucleotide introduced plant plant (monocotyledons or dicotyledons) for transforming or vegetable cell is changed.The appropriate method of described polynucleotide transformed plant cells is comprised: microinjection, electroporation, Agrobacterium-medialed transformation, direct gene transfer and high velocity ballistic bombardment etc.In certain embodiments, can utilize multiple transient transformation methods that ZmZIP4 gene of the present invention or ZmZIP6 gene are supplied to plant.In other embodiments, ZmZIP4 gene of the present invention or ZmZIP6 gene are incorporated in plant by being contacted with virus or viral nucleic acid by plant, usually, such method relates to be introduced ZmZIP4 gene of the present invention or ZmZIP6 gene construct in viral DNA or RNA molecule.

The cell regeneration stable conversion plant (McCormick et al.Plant Cell Reports.1986.5:81-84) utilizing ordinary method can make to have transformed.The present invention can be used for transforming any floristics, includes but not limited to: monocotyledons or dicotyledons; Preferably, described target plant comprises food crop, vegetables or fruit tree etc., is more preferably food crop, such as, can be the food crop such as corn, paddy rice, Resistance In Wheat And Barley, Chinese sorghum, soybean, potato.

The term definition arrived involved in the present invention

Unless otherwise defined, otherwise all technology used herein and scientific terminology all have with those skilled in the art usually understand identical implication.Although any method, device and the material similar or equivalent with person described herein can be used in practice of the present invention or test, preferred method, device and material are described now.

Term " recombinant host cell strain " or " host cell " mean the cell comprising polynucleotide of the present invention, and no matter use which kind of method to carry out inserting to produce recombinant host cell, such as directly absorb, transduce, known other method in f pairing or affiliated field.Exogenous polynucleotide can remain the non-integrated vector of such as plasmid or can be integrated in host genome.Host cell can be prokaryotic cell prokaryocyte or eukaryotic cell, and host cell also can be unifacial leaf or dicotyledonous plant cells.

Term " Nucleotide " means the deoxyribonucleotide of sub-thread or bifilar form, dezyribonucleoside, ribonucleoside or ribonucleotide and polymkeric substance thereof.Except nonspecific restriction, otherwise the nucleic acid of the known analogue containing natural nucleotide contained in described term, and described analogue has the binding characteristic that is similar to reference nucleic acid and carries out metabolism in the mode of the Nucleotide being similar to natural generation.Unless other specific restriction, otherwise described term also means oligonucleotide analogs, and it comprises PNA (peptide nucleic acid(PNA)), DNA analogue used in antisense technology (thiophosphatephosphorothioate, phosphamide acid esters etc.).Unless otherwise, otherwise the specific nucleic acid sequence sequence that also impliedly contains its conservative varient (including, but is not limited to degenerate codon replace) of modifying and complementary sequence and clearly specify.Particularly, the 3rd sequence replaced through mixing base and/or deoxyinosine residue by producing one of them or more than one selected (or all) codon replaces to realize degenerate codon (people such as Batzer, Nucleic Acid Res.19:5081 (1991); The people such as Ohtsuka, J.Biol.Chem.260:2605-2608 (1985); With people such as Cassol, (1992); The people such as Rossolini, Mol Cell.Probes8:91-98 (1994)).

Term " polypeptide ", " peptide " and " protein " exchange in this article and use with the polymkeric substance meaning amino-acid residue.That is, the description for polypeptide is equally applicable to describe peptide and describe albumen, and vice versa.It is the aminoacid polymers of non-naturally encoded amino acids that described term is applicable to natural generation aminoacid polymers and one of them or more than one amino-acid residue.As used herein, the amino acid chain of any length contained in described term, and it comprises full-length proteins (i.e. antigen), and wherein amino-acid residue connects via covalent peptide bonds.

Accompanying drawing explanation

The schematic diagram of Fig. 1 Yeast expression carrier pFL61.

The expression pattern of ZmZIP4, ZmZIP6 under Fig. 2 standard Hoagland culture medium condition; S (shoot), R (root).

Fig. 3 ZmZIP4 gene, the expression pattern of ZmZIP6 gene under various treatment condition.

Fig. 4 ZmZIP4 gene, the ZmZIP6 gene expression pattern in corn Embryo and endosperm development process.

The Phylogenetic tree analysis of the ZIP member of Fig. 5 different plant species.

Fig. 6 pRTL2NGFP-ZmZIP4, pRTL2NGFP-ZmZIP6 recombinant vectors enzyme cuts qualification.

Fig. 7 pRTL2NGFP-ZmZIP4 vector construction schema.

Fig. 8 pRTL2NGFP-ZmZIP6 vector construction schema.

Subcellular Localization in Fig. 9 ZmZIP4 and ZmZIP6 onion epidermis cell; GFP is pRTL2NGFP empty carrier positioning scenarios; ZmZIP4 is the Subcellular Localization situation of pRTL2NGFP-ZmZIP4; ZmZIP6 is the Subcellular Localization situation of pRTL2NGFP-ZmZIP6.

Subcellular Localization in Figure 10 ZmZIP4, ZmZIP6 Arabidopis thaliana mesophyll protoplast; GFP is pRTL2NGFP empty carrier positioning scenarios; ZmZIP4 is the Subcellular Localization situation of pRTL2NGFP-ZmZIP4; ZmZIP6 is the Subcellular Localization situation of pRTL2NGFP-ZmZIP6.

Figure 11 pFL61-ZmZIP4, pFL61-ZmZIP6 and pFL61-OsZIP5, pFL61-OsZIP8, pFL61-OsIRT1 forward connect recombinant vectors enzyme and cut qualification; M is the Marker of 1Kb; 1-5 is followed successively by pFL61-ZmZIP4, pFL61-ZmZIP6, pFL61-OsZIP5, pFL61-OsZIP8, pFL61-OsIRT1 double digestion result.

Figure 12 pFL61-ZmZIP4 vector construction schema.

Figure 13 pFL61-ZmZIP6 vector construction schema.

Figure 14 ZmZIP4, ZmZIP6 yeast complementation experiment result.

Figure 15 pCAMBIA3301-ZmZIP4 plant expression vector schematic diagram.

Figure 16 pBI121-ZmZIP6 plant expression vector schematic diagram.

Figure 17 ZmZIP4 gene process LAN in corn improves the experimental result of Zn content in corn; ZmZIP4 is the result turning iron and zinc-content determination in ZmZIP4 gene corn seed; Zheng58 is the result of zinc-content determination in non-transgenic zheng58 corn variety seeds.

Figure 18 ZmZIP6 gene process LAN in Arabidopis thaliana improves the experimental result of Zn content in Arabidopis thaliana; ZmZIP6 is the result turning iron and zinc-content determination in ZmZIP6 gene Arabidopis thaliana seed; WT is the result of iron and zinc-content determination in wild-type Colombia seed.

Embodiment

Further describe the present invention below in conjunction with specific embodiment, advantage and disadvantage of the present invention will be more clear along with description.But these embodiments are only exemplary, do not form any restriction to scope of the present invention.It will be understood by those skilled in the art that and can modify to the details of technical solution of the present invention and form or replace down without departing from the spirit and scope of the present invention, but these amendments and replacement all fall within the scope of protection of the present invention.

Experiment material

1.1 vegetable material

Corn inbred line X178 is provided by agricultural college of Agricultural University Of Hebei/laboratory, branch center, Hebei, national corn improvement center, and paddy rice self-mating system Japan is fine is so kind as to give by Beijing Normal University's school of life and health sciences.

1.2 bacterial strains and carrier

Intestinal bacteria (E.coli) bacterial strain Mach1-T1 and Agrobacterium (A.tumefacterium) bacterial strain EHA105, GV3101 preserve by this laboratory.PGEM-Teasy carrier is purchased from Promega company.Yeast expression carrier pFL61 (Fig. 1 is shown in by schematic diagram), yeast strain zrt1zrt2ZHY3 (MAT α ade6can1his3leu2trp1ura3zrt1::LEU2zrt2::HIS3), fet3fet4DEY1453 (MATa/MATa ade2/+can1/can1his3/his3leu2/leu2trp1/trp1ura3/ura3fet3-2::HIS3/fet3-2::HIS3fet4-1::LEU2/fet4-1::LEU2), DY1455 (MATa ade6can1his3leu2trp1ura3) teach friendship by Agricultural University Of Nanjing Zhang Hongsheng and are so kind as to give.

The clone of embodiment 1 corn zinc-iron regulation and control transporter ZmZIP4 gene and ZmZIP6 gene

1, the process of vegetable material

First vermiculite Hoagland nutritive medium is soaked into, corn inbred line X178 Seed Points is sowed in seedling pan, cover the dry vermiculite of last layer above, in greenhouse, (16h illumination/8h is dark, 26 DEG C) middle cultivation, 12 days seedling grow to 2 leaves wholeheartedly time move into growth in standard Hoagland nutritive medium and wholeheartedly (within every 3 days, change one time of nutrition liquid) to 3 leaves in 6 days, 3 leaves corn seedling is wholeheartedly at standard liquid nutrient and not zincification, iron, copper, manganese, Gao Xin, 0 is processed under the condition of iron, 6, 12, 24, 48, after 96h, collect seedling overground part and root respectively, preserve in-80 DEG C after liquid nitrogen flash freezer and be used for Total RNAs extraction.

2, the extraction of corn total serum IgE

Adopt Trizol to extract test kit and extract corn total serum IgE.

3, the synthesis of cDNA

(1), DNA is removed, by following preparation reaction system:

Total serum IgE (1 μ g/ μ L) 1.0 μ L, DNAse I (10U/ μ L) 1.0 μ L, 10 × DNAse I buffer1.0 μ L, DEPC H ₂o7.0 μ L, amounts to 10.0 μ L; 37 DEG C of 30min, add 1 μ L25mM EDTA, 65 DEG C of 5min termination reactions.

(2), 1 μ L oligo (dT18) is added, 65 DEG C of 5min;

(3), totally 12 μ L above, add following component again and obtain reverse transcription system: 5 × reaction buffer 4.0 μ L, Ri RT (20U/ μ L) 1.0 μ L, Re RT (200U/ μ L) 1.0 μ L, 10mM dNTP mix2.0 μ L, amounts to: 20.0 μ L; 42 DEG C of 60min, 70 DEG C of 5min, termination reaction.

4, the clone of goal gene

(1), according to the ORF frame design primer of goal gene:

ZmZIP4F5'-ATGGACGCCACGCGAGTTCG-3'

ZmZIP4R5'-CTACGCCCATTTGGCGAGCAAC-3'

ZmZIP6F5'- TACGTAATGTCCGGCACCGGGT-3'SnaBI

ZmZIP6R5'- TACGTACTATGCCCAGAGAGCTAATACCG-3'SnaBI

With the cDNA of above-mentioned steps 3 for template, select ExTaq enzyme, 2 × GCI buffer carries out pcr amplification, and PCR program is: 95 DEG C of denaturation 4min; 94 DEG C of sex change 1min, 60 DEG C of annealing 1min, 72 DEG C extend 1min, 33 circulations; 72 DEG C extend 10min;

(2), being cloned in pGEM-T carrier by cloning the fragment obtained, transforming Mach1-T1 bacterial strain;

(3), cut qualification through enzyme and obtain positive recombinant plasmid called after pGEM-ZmZIP4 and pGEM-ZmZIP6 respectively, order-checking is correctly cloned, one of them unnamed gene of cloning is ZmZIP4, the cDNA sequence of this gene is for shown in SEQ ID No.1, and the aminoacid sequence of deriving is for shown in SEQ ID No.2; Another unnamed gene of cloning is ZmZIP6, and the cDNA sequence of this gene is for shown in SEQ ID No.3, and the aminoacid sequence of deriving is for shown in SEQ ID No.4.

The expression pattern of embodiment 2 ZmZIP4 and ZmZIP6 in seedling, Fetal liver cells

The cDNA of reverse transcription in embodiment 1 step 3 is diluted 10 times of templates as PCR reaction, PCR reaction system is as follows:

CDNA2.0 μ L, ExTaq0.1 μ L, 2 × GCI damping fluid 10.0 μ L, 10mM dNTP mix0.8 μ L, upstream primer RTZmZIP4F/RTZmZIP6F (10 μMs/μ L) 1.0 μ L, downstream primer RTZmZIP4R/RTZmZIP6R (10 μMs/μ L) 1.0 μ L, ddH ₂o5.1 μ L, amounts to 20.0 μ L;

RTZmZIP4F5'-CCTTCTTCTCGCTCACCGCT-3'

RTZmZIP4R5'-AGCCTCGGGTTGCTGAAGT-3'

RTZmZIP6F5'-CATCGCACAGGCTGGTTTTG-3'

RTZmZIP6R5'-TTCCAGCCGAAAGTGAACCA-3'

PCR reaction conditions: 94 DEG C of denaturation 4min; 30 circulations, each circulation 94 DEG C of sex change 45 seconds, 60 DEG C of annealing 1min, 72 DEG C extend 1min; Finally extend 72 DEG C of 10min again, be cooled to 16 DEG C, take out PCR primer and put into 4 DEG C of preservations.

The detection of destination gene expression amount: in embodiment 1 step 3, the cDNA of reverse transcription dilutes 20 times of templates of reacting as Real-time PCR, and Actin is internal reference, and reaction system is as follows:

CDNA5.0 μ L, SYBR Green I10.0 μ L, Rox0.4 μ L, upstream primer ZmActin1F (10 μMs/μ L) 0.4 μ L, downstream primer ZmActin1R (10 μMs/μ L) 0.4 μ L, ddH ₂o3.8 μ L, amounts to 10.0 μ L;

ZmActin1F5'-ATGTTTCCTGGGATTGCCGAT-3'

ZmActin1R5'-CCAGTTTCGTCATACTCTCCCTTG-3'

Program thereby: 95 DEG C of 2min, 95 DEG C of 15sec, 60 DEG C of 34sec, 40 circulations, by Δ Δ Ct method calculation expression amount.

Found by real-time RT-PCR expression analysis, under normal nutrition condition, ZmZIP4, the expression amount higher than underground part (Fig. 2) in ZmZIP6 portion on the ground, under high zink rod part, the expression amount of ZmZIP4 portion and underground part on the ground increases gradually, under high ferro condition, the expression amount of ZmZIP4 portion and underground part on the ground presents the trend raised gradually along with the prolongation of time, 96h reaches the highest, but, no matter ZmZIP6 is under high zinc or high ferro condition, the expression amount of overground part and underground part does not all significantly change (Fig. 3), these results show, ZmZIP4 seedling period the concentration to zinc and iron more responsive and ZmZIP6 is insensitive to zinc-iron concentration.After pollination different number of days Fetal liver cells in expression analysis find, ZmZIP4 expression amount in the embryo of 17 days after pollination raises, and the expression amount of ZmZIP6 in the pollination embryo of latter 21 days reaches the highest (Fig. 4).Infer that ZmZIP4 may play a role in the growth course of embryo, ZmZIP6 may be relevant with the maturation of embryo.

The bioinformatic analysis of embodiment 3 ZmZIP4 and ZmZIP6

ZmZIP4 and ZmZIP6 gene is positioned on the 4th and the 8th karyomit(e) of corn respectively, and ZmZIP4 is made up of 3 exons and 2 introns, and ZmZIP6 is made up of 2 exons 1 introns.ZmZIP4 encodes 386 amino acid, ZmZIP6 genes encoding 396 amino acid, by finding the amino acid alignment of corn ZIP albumen, ZmZIP4 albumen contains 6 membrane spaning domains, ZmZIP6 albumen contains 8 membrane spaning domains, the variable region that one is rich in Histidine is had between the 3rd and the 4th cross-film district, may be relevant with the binding transport of metal ion.Phylogenetic analysis shows, and ZmZIP4 and OsZIP4 is in a branch, and ZmZIP6 and OsZIP6 is in a branch (Fig. 5), and OsZIP4 is a zinc transporter, so ZmZIP4 and ZmZIP6 may be zinc-iron transporter.

The Subcellular Localization of albumen coded by embodiment 4 ZmZIP4 and ZmZIP6

1, the structure of fusion expression vector

According to the primers of ZmZIP4 and ZmZIP6 gene, primer sequence is as follows:

ZmZIP4GF5'- CTCGAG?ATGGACGCCACGCGAGTTCG-3'XhoI

ZmZIP4GR5'- TCTAGACGCCCATTTGGCGAGCAAC-3'XbaI

ZmZIP6GF5'- CCATGGCGATGTCCGGCACCGGGTG-3'NcoI

ZmZIP6GR5'- TCTAGATGCCCAGAGAGCTAATACCGACAT-3'XbaI

Add suitable restriction enzyme site, and gene 3 ' is held and is removed terminator codon, check order correct plasmid for template to be connected to during clone gene on pGEM-T carrier, and select ExTaq enzyme and 2 × GCI buffer to carry out pcr amplification, PCR program is: 95 DEG C of denaturation 4min; 94 DEG C of sex change 1min, 60 DEG C of annealing 1min, 72 DEG C extend 1min, 33 circulations; 72 DEG C extend 10min.Amplified fragments reclaims rear clone in pGEM-T carrier through 1% agarose gel electrophoresis, transform Escherichia coli strain Mach1-T1, and obtain positive colony through LB substratum (IPTG, X-gal, Amp), upgrading grain, enzyme are cut and sequence verification; Check order correct plasmid for template to be connected to during clone gene on pGEM-T carrier, ExTaq enzyme and 2 × GCI buffer is selected to carry out pcr amplification, amplified fragments reclaims rear clone in pGEM-T carrier through 1% agarose gel electrophoresis, check order after correct plasmid enzyme restriction, object fragment is building up on pRTL2NGFP carrier, called after pRTL2NGFP-ZmZIP4 or pRTL2NGFP-ZmZIP6, Fig. 6 are that enzyme cuts qualification figure, Fig. 7 and Fig. 8 is for building schema.

2, with corresponding enzyme cut pRTL2NGFP carrier cut from different enzymes after gene, through T ₄dNA ligase connects, and transforms Mach1-T1 bacterial strain, carries plasmid enzyme restriction evaluation and screening and go out the large upgrading grain of correct recombinant chou for via Particle Bombardment Transformation onion epidermis.

3, the preparation of the micro-bullet of particle gun

4, onion epidermis conversion is carried out with particle gun

(Fig. 9) plasma membrane and intercellular membrane is positioned at from positioning result known ZmZIP4, ZmZIP6.For determining the concrete location of intercellular membrane further, select ER marker and ZmZIP4, ZmZIP6 to locate the conversion carrying out Arabidopis thaliana mesophyll protoplast altogether, result proves that ZmZIP4, ZmZIP6 are positioned at (Figure 10) in cytoplasmic membrane and endoplasmic reticulum.

Embodiment five yeast complementation experiment

1, the structure of Yeast expression carrier

Suitable restriction enzyme site design primer is added according to goal gene sequence:

ZmZIP4YF5'- TACGTAATGGACGCCACGCGAGTTCG-3'SnaBI

ZmZIP4YR5'- TACGTACTACGCCCATTTGGCGAGCAAC-3'SnaBI

ZmZIP6YF5'- TACGTAATGTCCGGCACCGGGT-3'SnaBI

ZmZIP6YR5'- TACGTACTATGCCCAGAGAGCTAATACCG-3'SnaBI

OsZIP5YF5'- CCCGGGGAGCCATCGGCGATGGCGA-3'SmaI

OsZIP5YR5'- GAGCTCGTGATGGTCACTCACTCATCACGCC-3'SacI

OsZIP8YF5'- GCGGCCGCATGAGGACGAACACCACC-3'NotI

OsZIP8YR5'- GCGGCCGCCCTCTACATTAGTCCCTGAG-3'NotI

OsIRT1YF5'- GCGGCCGCCCCGGGATGGCGACGCCGCGGA-3'NotI,SmaI

OsIRT1YR5'- GCGGCCGCCCCGGGTCACGCCCACTTGGCCATG-3'NotI,SmaI

Check order correct plasmid for template to be connected to during clone gene on pGEM-T carrier, and select ExTaq and 2 × GCI buffer to carry out pcr amplification, PCR program is: 95 DEG C of denaturation 4min; 94 DEG C of sex change 1min, 60 DEG C of annealing 1min, 72 DEG C extend 1min, 33 circulations; 72 DEG C extend 10min.Amplified fragments reclaims rear clone in pGEM-T carrier through 1% agarose gel electrophoresis, transform Escherichia coli strain Mach1-T1, positive colony is obtained through LB substratum (IPTG, X-gal, Amp), upgrading grain, enzyme are cut and sequence verification, check order after correct plasmid enzyme restriction, object fragment is building up on pFL61 carrier, called after pFL61-ZmZIP4, pFL61-ZmZIP6 and pFL61-OsZIP5, pFL61-OsZIP8 and pFL61-OsIRT1, Figure 11 is that enzyme cuts qualification figure, Figure 12 and Figure 13 is for building schema.With NotI enzyme cut pFL61 carrier and enzyme cut after ZmZIP4 or ZmZIP6 gene through T ₄dNA ligase connects, and transforms Mach1-T1 bacterial strain, carries plasmid enzyme restriction evaluation and screening and go out the large upgrading grain of correct recombinant chou for transformed saccharomyces cerevisiae.

2, electroporated method transformed yeast

(1), from single bacterium colony of picking zrt1zrt2ZHY3, fet3fet4DEY1453 and DY1455 YPD flat board in the YPD liquid nutrient medium of 20mL, about 24h cultivated by 28 DEG C of shaking tables;

(2), the bacterium liquid of drawing above 2% volume is transferred in the YPD substratum of 100mL and continues to expand numerous about 4-5h, can prepare competence when bacterium liquid OD600 is 1.2-1.5;

(3), bacterium liquid is collected in the centrifuge tube of 50mL, 4 DEG C, 5,000rpm, centrifugal 5min, outwells supernatant;

(4), add isopyknic deionized water, on ice resuspended thalline, 4 DEG C, 5,000rpm, 5min are centrifugal, outwell supernatant;

(5), add the deionized water of 1/2 volume, resuspended thalline on ice, 4 DEG C, 5,000rpm, 5min are centrifugal, outwell supernatant;

(6), add the 1M Sorbitol Solution USP of 10mL, resuspended thalline on ice, 4 DEG C, 5,000rpm, 5min are centrifugal, outwell supernatant;

(7), add the Sorbitol Solution USP of 450-600 μ L, gently inhale with the rifle head of decaptitating, resuspended thalline;

(8) competence adding about 100 μ L in the centrifuge tube, according to each 1.5mL is as the criterion, packing;

(9), in every pipe competence add appropriate DNA (10 μ about L, c >=200ng/ μ L), place 1-2min on ice, be drawn onto afterwards in the electric shock cup of precooling, do not have bubble;

(10), electroporated, add about 800 μ L immediately, the Sorbitol Solution USP of the precooling of 1M, resuspended thalline;

(11), from electric shock cup sucking-off thalline, SD/Ura-is dull and stereotyped in coating;

(12), the dull and stereotyped upper 28 DEG C of cultivations of SD can grow macroscopic bacterial plaque in about 6 days.

3, the qualification of yeast-positive clone

(1), get 1.5mL yeast culture, centrifugal 30 seconds of 9,000rpm, inhale as much as possible and abandon supernatant, collect yeast cell;

(2), add 600 μ L Sorbitol buffer, the abundant re-suspended cell of soft piping and druming, adds the Lyticase of 80U, fully puts upside down mixing, 37 DEG C of incubation 30min peptic cell walls, and centre is put upside down for several times;

(3), 13,000rpm centrifugal 1min, inhale as far as possible and abandon supernatant, add the resuspended bacterial sediment of 250 μ L solution YP1, vortex concussion is to thoroughly suspending;

(4), add 250 μ L YP2 solution, gently overturn, make the abundant cracking of thalline, room temperature places 4min;

(5), add 350 μ L YP3 solution, gently overturn, fully there will be white flock precipitate during mixing, leave standstill the centrifugal 5min of 3-5min, 13,000rpm on ice, careful Aspirate supernatant.

(6), by previous step gained supernatant liquor add (adsorption column puts into collection tube) in adsorption column AC, 12,000rpm centrifugal 30-60 second, outwell the waste liquid in collection tube;

(7), add 500 μ L protein liquid removal PD, 12,000rpm centrifugal 30-60 second, abandon waste liquid;

(8), add 500 μ L rinsing liquids WB (adding dehydrated alcohol), 12,000rpm centrifugal 30-60 second, abandon waste liquid;

(9), add 500 μ L rinsing liquid WB, 12,000rpm centrifugal 30-60 second, abandon waste liquid;

(10), by adsorption column AC put back in sky collection tube, the centrifugal 2min of 13,000rpm, removing rinsing liquid;

(11), take out adsorption column AC, put into a clean centrifuge tube, add 50 μ L elution buffer EB (65-70 DEG C of water-bath) in the middle part of adsorption film, room temperature places the centrifugal 1min of 2min, 13,000rpm.

(12), with 1 μ L DNA of extracting for template, the two ends primer of gene is pcr amplification primer, carries out pcr amplification checking goal gene, verifies correct bacterium liquid, add the glycerine of 25% in-80 DEG C of preservations.

4, yeast complementation experiment result

PFL61, pFL61-ZmZIP4, pFL-ZmZIP6, pFL61-OsZIP5, pFL61-OsZIP8, pFL61-OsIRT1 plasmid is transformed into respectively in yeast mutant zrt1zrt2ZHY3 and fet3fet4DEY1453, pFL61 is negative control, OsZIP5, OsZIP8 (Lee S, Kim SA, Lee J, et al.Zinc deficiency-inducible OsZIP8encodes a 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 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 experimental botany2007, 58 (11): 2909-2915.) be the positive control of zinc transporter, OsIRT1 (Lee S, An G.Over-expression of OsIRT1leads to increased iron and zinc accumulations in rice.Plant, cell & environment2009, 32 (4): 408-416.) be the positive control of iron transporters, pFL61 transformed wild type bacterial strain DY1455 is as another positive control, positive yeast is accredited as after conversion, cultivate in SD liquid nutrient medium, yeast liquid dilutes 4 concentration (OD respectively ₆₀₀=1,0.1,0.01,0.001), then get 5 μ L points in the substratum of low zinc, low iron and normal SD, (SD substratum adds 0.4mM EDTA, 0.4mM EDTA and 250 μMs of ZnSO to low zinc substratum ₄, 0.4mM EDTA and 300 μM ZnSO ₄), (SD substratum adds 50mM MES, 50mM MES and 50 μMs of FeCl to low iron substratum ₃, 50mM MES and 100 μM FeCl ₃) yeast complementation reference Lin, test (the Lin YF of Y.F, Liang HM, Yang SY, et al.Arabidopsis IRT3is a zinc-regulated and plasma membrane localized zinc/iron transporter.The New phytologist2009,182 (2): 392-404.) carry out, 28 DEG C of cultivations, 6 days viewing test results.

Yeast complementation result shows, and under low zink rod part, is added with 250 μMs of ZnSO ₄substratum in obviously can observe DY-pFL61 (wild-type), Z-ZmZIP4, Z-ZmZIP6, Z-OsZIP5, Z-OsZIP8, Z-OsIRT1 are better than empty carrier Z-pFL61 growing way, further, ZmZIP4, ZmZIP6 are suitable with the rice Os ZIP growing way reported.Under low iron bar part, D-ZmZIP4, D-ZmZIP6 are better than empty carrier D-pFL61 growing way, but the transport activity of the D-OsIRT1 do not reported strong (Figure 14), no matter ZmZIP4, ZmZIP6 are at low zinc or the transport activity shown under low iron bar part in various degree, illustrate that ZmZIP4, ZmZIP6 have the function absorbing or transport zinc-iron in plant materials.

Experimental example 1 ZmZIP4 gene and ZmZIP6 gene respectively in corn and Arabidopis thaliana process LAN improve the experiment of iron and Zn content in corn and Arabidopis thaliana seed

The structure that is connected by plant expression vector pCAMBIA3301 and pBI121 that ZmZIP4 gene, ZmZIP6 gene control with composing type 35S promoter respectively obtains ZmZIP4 genetically modified plants expression vector and ZmZIP6 gene plant expression vector (Figure 15, Figure 16); Be transformed in corn or Arabidopis thaliana by the recombinant plant expression vector of structure, qualification obtains positive turning ZmZIP4 gene corn and turn ZmZIP6 gene Arabidopis thaliana; By the positive turn ZmZIP4 gene corn and turn ZmZIP6 gene Arabidopis thaliana and non-transgenic corn and wild-type Colombia Arabidopis thaliana identical carry training condition under cultivate, results turn ZmZIP4 gene corn seed, turn ZmZIP6 gene Arabidopis thaliana seed and non-transgenic corn seed and Arabidopis thaliana seed, measure respectively to turn ZmZIP4 gene corn seed, turn the content of iron or zinc in ZmZIP6 gene Arabidopis thaliana seed, non-transgenic zheng58 corn seed and wildtype Arabidopsis thaliana seed; Take a certain amount of seed material through micro-wave digestion, constant volume, carry out the mensuration of zinc iron content by ICP-MS method; Often criticize and measure 200mg seed powder, measure three batches, get the mean value of three batch datas.Measurement result is shown in Figure 17 and Figure 18.From the result of Figure 17 and Figure 18, in plant materials, process LAN ZmZIP4 gene or ZmZIP6 gene can iron or Zn content in raising plant seed in various degree.

Claims

1. the zinc-iron regulation and control transporter ZmZIP4 gene be separated from corn (Zea mays), it is characterized in that, its cDNA sequence is selected from (a), (b) or (c):

Nucleotide sequence shown in (a) SEQ ID No.1;

Amino acid whose nucleotide sequence shown in (b) coding SEQ ID No.2;

2. the zinc-iron regulation and control transporter ZmZIP6 gene be separated from corn (Zea mays), it is characterized in that, its cDNA sequence is selected from (a), (b) or (c):

Nucleotide sequence shown in (a) SEQ ID No.3;

Amino acid whose nucleotide sequence shown in (b) coding SEQ ID No.4;

3. the protein of genes encoding described in claim 1 or 2.

4. according to protein according to claim 3, it is characterized in that, the aminoacid sequence of described protein is for shown in (a) or (b):

(a) SEQ ID No.2 or the aminoacid sequence shown in SEQ ID No.4;

(b) by the aminoacid sequence shown in SEQ ID No.2 or SEQ ID No.4 by the replacement of one or more amino-acid residue, disappearance or/and insert and the derivative protein variant still with zinc-iron regulation and control transporter function obtained.

5. the recombinant expression vector containing zinc-iron regulation and control transporter gene described in claim 1 or 2; Preferably, described recombinant expression vector is recombinant plant expression vector.

6. zinc-iron regulation and control transporter gene described in claim 1 or 2 is in the application regulated and controled or improve plant absorption, transhipment or store in zinc or iron ability.

7. the application of zinc-iron regulation and control transporter gene described in claim 1 or 2 in the application promoted or regulate and control in radicle and embryonic development or promotion embryo of plant seed maturation.

8. described in claim 1 or 2, zinc-iron regulation and control transporter gene is increasing the application in food crop seed zinc or iron level.

9. the application of zinc-iron regulation and control transporter gene in the excessive zinc of releasing or iron are poisoned plant materials described in claim 1 or 2.

10. according to the application of claim 6-9 described in any one, it is characterized in that, comprising: transporter gene is exercisable is connected with expression regulation element by zinc-iron regulation and control described in claim 1 or 2, obtains recombinant plant expression vector; By recombinant plant expression vector transformation receptor plant or vegetable cell, cultivate and obtain transgenic plant.