CN104212815B - Zea mays zinc iron-regulated transporter ZmIRT1 gene and its applications - Google Patents

Abstract

The invention discloses a Zea mays zinc iron-regulated transporter ZmIRT1 gene and its applications. The Zea mays zinc iron-regulated transporter ZmIRT1 gene is separated from Zea mays, and the cDNA sequence of the gene is represented by SEQ ID No.1. Subcellular localization shows that the zinc iron-regulated transporter is localized in the plasma membrane and endoplasmic reticulum of a cell, has zinc and iron absorption, transportation and storage effects in plant cells, and particles in detoxification of zinc and iron ions. Quantitative real-time RT-PCR expression analysis shows expression the expression of the ZmIRT1 in zinc or iron deficient root and overground parts rises, the expression in the later stage of embryo growth obviously rises, and the ZmIRT1 plays an important role in the regulation of the embryo growth. Yeast complementation experiments show that the ZmIRT1 gene has a zinc and iron complementation ability. The separated ZmIRT1 gene has important application prospects in the regulation of zinc and iron absorption, transportation and storage of plants, promotion of the embryo growth and increase of the content of zinc and iron in grains of food crops.

Description

Semen Maydiss zinc-iron regulation and control transporter ZmIRT1 gene and its application

Technical field

The present invention relates to from plant detached metal-ions transportation body gene, detached and zinc more particularly, to from Semen Maydiss The relevant regulation and control transporter ZmIRT1 gene of the absorption of ferrum, transhipment or storage, the invention further relates to this regulation and control transporter ZmIRT1 gene regulation and control plant absorption, transhipment or storage zinc or ferrum ability, release excessive zinc-iron to the murder by poisoning of plant and Increase the application in cereal crops seed zinc iron content, belong to separation and the application neck that plant metal ion regulates and controls transporter gene Domain.

Background technology

Zinc and ferrum are trace element necessary to organism, important role in the growth and development process of plant (Wintz H,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 chemistry 2003,278(48):47644-47653.).Zinc It is structure cofactor (Haydon MJ, the Cobbett CS.A novel of more than 300 kind of enzyme of organism and key protein major facilitator superfamily protein at the tonoplast influences zinc tolerance and accumulation in Arabidopsis.Plant physiology 2007,143(4):1705- 1719.).Zinc is not only involved in the various metabolism of body, biomembrane is stable and the physiological function such as gene expression regulation in also undertake 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 lead to the Oxidative demage of chlorophyll, lipid, albumen, plasma membrane, increases plant in right amount The content of interior zinc can improve crop yield, and in plant body, building up of zinc ion can produce murder by poisoning to plant.

Ferrum plays a significant role in the catalytic reaction process of Cellular respiration, photosynthesis and metalloprotein, is important Electron transit mediator, therefore, ferrum element has irreplaceable function in protokaryon and Eukaryotic vital movement.In addition, it is thin The too high Fe of intracellular³⁺/Fe²⁺Oxidation-reduction potential can lead to 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 vie 1999,322(1):43-54.).Therefore, strictly control plant The balance of interior metal ion it is critical that.

The albumen participating in zinc-iron absorption mainly has three classes, is all to be existed with protein family form, including：ZIP, i.e. zinc tune Control transporter (Zinc-regulated transporter, ZRT) and ferrum regulation and control transporter (Iron-regulated Transporter, IRT).Yeast function complementation experiment display ZIP family gene can be transported including Zn²⁺、Fe²⁺、Cu²⁺、Cd²⁺ In 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 biology 2006, 9(3):322-330.).ZIP is typically made up of 309-476 amino acid residue, has 8 potential membrane spaning domains and similar Topological structure, has a long variable region between the 3rd and the 4th transmembrane region, variable region is located at intracellular, and its C, N-terminal, positioned at extracellular, are somebody's turn to do Area is rich in histidine residues, may (Guerinot ML.The ZIP family of relevant with the combination of metal, transhipment metal transporters.Biochim Biophys Acta 2000,1465(1-2):190-198.).

Identify ZIP in the plants such as arabidopsiss, Oryza sativa L., M. truncatula, Semen sojae atricolor, wild type emmer wheat, Fructus Vitis viniferae at present Gene is simultaneously studied to its function.Find 16 ZIP family genes in arabidopsiss, AtIRT1 is real by yeast complementation Test and separate the first ZIP functional gene obtaining, it is mainly expressed in root, and the overexpression of this gene may result in the excessive of nickel Accumulation (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 metal transporter gene IRT1results in iron deficiency accompanied by cell differentiation defects.Plant molecular biology 2002,50(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 biology 2002,31(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 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 accumulation in Arabidopsis thaliana.Plant&cell physiology 2011,52(8):1433-1442.).AtIRT2 mainly expresses in root, fixed Position is in vesicle thus it is speculated that having function of detoxification (Vert G, the Briat JF, Curie of intracellular excessive metallic element C.Arabidopsis IRT2gene encodes a root-periphery iron transporter.The Plant journal: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 uptake system to maintain iron homeostasis in root epidermal cells.Planta 2009,229 (6):1171-1179.14,15).AtIRT3 energy mutually zinc supplement, iron transfer double-mutant, overexpression AtIRT3 can make zinc on the ground Portion, ferrum accumulate (Lin YF, Liang HM, Yang SY, et al.Arabidopsis IRT3is a zinc- in underground part regulated and plasma membrane localized zinc/iron transporter.The New phytologist 2009,182(2):392-404.).Expression analysis show, AtZIP1, AtZIP5, AtZIP9, AtZIP12 and AtIRT3 is induced by zinc deficiency, thus speculates, these genes under the conditions of zinc deficiency may strengthen zinc absorbability (Kramer U, Talke IN,Hanikenne M.Transition metal transport.FEBS Lett 2007,581(12):2263- 2272.).

Semen Maydiss (Zea mays) are the important grain of China, feedstuff and industrial crops, increase zinc, ferrum etc. in corn kernel micro- The content of secondary element, to improving diet or food utilization efficiency, promote the development of economy and health is particularly important.At present, The transport protein of known many take part in zinc-iron ionic equilibrium network system in plant body, wherein ZIP (Zinc-regulated Transporters, Iron-regulated transporter-like proteins, ZIP) gene family is to zinc, ferrum etc. two The absorption of valence metal ion, transport and storage play an important role, and on arabidopsiss, Oryza sativa L., Fructus Hordei Vulgaris, Semen sojae atricolor, have reported one A bit about the research of ZIP family gene, but in plant body, specific mechanism of action not yet understands completely to ZIP family gene, And the research report with regard to the ZIP family gene of Semen Maydiss is less.Understand Zn²⁺、Fe²⁺Absorption in Semen Maydiss, means of transportation, point Cloth rule and regulatory mechanism, it will help improve growth promoter in zinc, sideropenic environment for the Semen Maydiss, for disclosing jade further In rice, the mechanism of action of ZIP family gene lays the foundation, and provides candidate gene for corn zinc, ferrum high-efficient transgenic breeding, also for Mankind's zinc-iron nutrition provides good basis.

Content of the invention

An object of the present invention is to provide detached zinc-iron from Semen Maydiss (Zea mays) to regulate and control transporter gene；

The second object of the present invention is the protein providing zinc-iron to regulate and control coded by transporter gene；

The third object of the present invention is to be applied to plant to gold such as zinc-irons by described zinc-iron regulation and control transporter gene Belong to absorption, transhipment or the storage of ion, releasing excessive zinc-iron to the murder by poisoning of plant or increases zinc-iron in cereal crops seed Content.

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

From Semen Maydiss (Zea mays), detached zinc-iron regulates and controls transporter ZmIRT1 gene, and its cDNA sequence is (a), (b) Or shown in (c):

Nucleotide shown in (a), SEQ ID No.1；

(b), the nucleotide of aminoacid shown in coding SEQ ID No.2；

(c) and SEQ ID NO:The nucleotide that 1 complementary seriess can be hybridized in stringent hybridisation conditions, this nucleoside Protein coded by acid has the function that zinc-iron regulates and controls transporter.

Described " stringent hybridisation conditions " mean the condition of known low ionic strength and high temperature in the art.Generally, Under high stringency conditions, probe and its target sequence hybridize can detection level than with other sequence hybridizations can detection level higher (for example exceed at least 2 times of background.Stringent hybridisation conditions are sequence dependent, will be different under different environmental conditions, relatively Long sequence specific hybrid at relatively high temperatures.Can be identified and probe by controlling the preciseness hybridizing or wash conditions The target sequence of 100% complementation.Detailed guidance for nucleic acid hybridization refers to relevant document (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 are typically selected to be less than distinguished sequence under regulation ionic strength pH About 5-10 DEG C of heat fusion joint (Tm).Tm be in the state of the equilibrium 50% probe complementary with target hybridize to residing during target sequence Temperature (under specified ionic strength, pH and nucleic acid concentration) (because target sequence is present in excess, in equilibrium-like under Tm Under state, 50% probe is occupied).High stringency conditions can be following condition：Wherein it is below about in the lower salinity of pH 7.0 to 8.3 1.0M Na ion concentration, typically about 0.01 arrives 1.0M Na ion concentration (or other salt), and temperature (includes for short probe 10 to 50 nucleotide of (but not limited to)) for be at least about 30 DEG C, and for long probe (including but not limited to be more than 50 Nucleotide) for be at least about 60 DEG C.High stringency conditions also can be realized by adding the destabilizing agent of such as Methanamide.For choosing For selecting property or specific hybrid, positive signal can be the background hybridization of at least twice, optionally for 10 times of background hybridizations.Exemplary Stringent hybridisation conditions can be as follows：50% Methanamide, 5 × SSC and 1%SDS, cultivate at 42 DEG C；Or 5 × SSC, 1%SDS, Cultivate at 65 DEG C, washing and washing in 0.1%SDS at 65 DEG C in 0.2 × SSC.Described washing can carry out 5,15,30, 60th, 120min or the longer time.

Preferably, the cDNA sequence that the separated zinc-iron from Semen Maydiss of the present invention regulates and controls transporter ZmIRT1 gene is SEQ Shown in ID No.1.

The two of the object of the invention are to provide to regulate and control can absorbing, turning of transporter ZmIRT1 coded by said gene by above-mentioned zinc-iron Fortune or the protein of storage zinc-iron, the aminoacid sequence of this protein is shown in (a) or (b):

Aminoacid shown in (a), SEQ ID No.2；

(b), by the aminoacid shown in SEQ ID No.2 by the replacement of one or more amino acid residues, disappearance or/and Insert and the derivative protein variant still with zinc-iron regulation and control transhipment body function obtaining；

Particularly preferred, the aminoacid sequence that zinc-iron of the present invention regulates and controls transporter is shown in SEQ ID No.2.

Described " multiple " generally mean that 2-8, preferably 2-4, and this depends on the three-dimensional knot of zinc-iron regulation and control transporter The species of the position of amino acid residue or aminoacid in structure；Described " replacement " refers to be replaced with different amino acid residues respectively One or more amino acid residues；Described " disappearance " refers to the minimizing of amino acid residue quantity, that is to say and lacks wherein respectively One or more amino acid residues；Described " insertion " refers to the change of amino acid residue sequence, relatively for natural molecule, Described change leads to add one or more amino acid residues.

Protein variant of the present invention can be produced by genetic polymorphism or manual operation, and these operational approach are usually this Field is understood.For example, amino acid sequence variation or the fragment that zinc-iron regulates and controls transporter can be prepared by the mutation of DNA, its In due to mutation or change polynucleotide method be this area known by.Wherein, conservative replacement is will be residual for a kind of aminoacid Base is substituted for another kind of aminoacid with similar quality.Therefore, zinc-iron regulation and control transporter of the present invention and its coding base Because including naturally occurring sequence and two kinds of forms of variant." variant " means the sequence of basic simlarity, for polynucleotide, variant Comprise disappearance, insertion or/and the replacement of the one or more nucleotide of one or more site in native polynucleotide.For many Nucleotide, conservative variant includes not changing those variants of the aminoacid sequence of coding due to the degeneracy of genetic code. Such naturally occurring variant can be identified by existing Protocols in Molecular Biology.Variant polynucleotides also include synthesizing The polynucleotide in source, for example with the many nucleoside still encoding the aminoacid shown in SEQ ID No.2 obtained by direct mutagenesises Sour variant, or by the method (such as DNA rearrangement etc.) of restructuring.Those skilled in the art can be by following molecular biosciences skill Art means are screening or to evaluate function or the activity of albumen coded by variant polynucleotides：DNA binding activity, the phase between albumen Interaction, instantaneous study in the effect expressed in the activation situation of gene expression or transgenic plant etc..

The present invention separates from Semen Maydiss, has cloned zinc-iron regulation and control transporter ZmIRT1 gene, the experiment knot of Subcellular Localization Fruit shows, ZmIRT1 gene coded protein is positioned at plasma membrane and endoplasmic reticulum, therefore speculates its removing toxic substances with zinc-iron ion and storage Relevant, plant cell plays absorption, transhipment and storage zinc-iron and participates in the functions such as the removing toxic substances of zinc-iron ion.In subcellular fraction On the basis of positioning, the present invention has carried out expression analysis to the corn germ and endosperm of different number of days after pollination further, and result is sent out Existing, ZmIRT1 expresses and all raises in the root of iron deficiency and overground part, and the aerial partss expression of zinc deficiency 96h increases, and illustrates, ZmIRT1 Gene has the function that regulation and control plant (including under ground portion and aerial partss) absorbs, transports zinc-iron；Additionally, ZmIRT1 gene master Embryo to be expressed, and, ZmIRT1 raises in the later stage experssion of embryonic development, illustrates that ZmIRT1 is relevant with the maturation of embryo, and joins Accumulation with zinc or iron content in regulation and control seed.

Yeast complementation experiment shows, the ZIP gene phase in the separated ZmIRT1 gene of the present invention and Oryza sativa L. and arabidopsiss Seemingly, there is zinc-iron transport function.

At present it is known that many transport proteins take part in zinc-iron ionic equilibrium network system in plant body, some albumen also by It is applied in the transgenic research of plant, such as in Fructus Hordei Vulgaris, overexpression AtZIP1 gene can increase zinc and ferrum in seed Content (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.), equally, overexpression OsIRT1 gene, in Oryza sativa L., the content of zinc and ferrum has all carried in portion, underground part and seed on the ground High (Lee S, An G.Over-expression of OsIRT1leads to increased iron and zinc accumulations in rice.Plant,cell&environment 2009,32(4):408-416.).However, in Oryza sativa L. Middle overexpression OsZIP4, OsZIP5, OsZIP8, OsZIP9 result leads to excessive zinc to be gathered in root, reduces plant above ground Partial Zn content (Lee S, Kim SA, Lee J, et al.Zinc deficiency-inducible OsZIP8encodes a 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 experimental botany 2007,58(11):2909-2915.) be not reaching in seed Increase the purpose of Zn content, therefore, the overexpression of these genes is unfavorable to the enrichment of zinc in rice grain.These result tables Bright, dystopy overexpression may play a role with distribution for the accumulation of zinc-iron.However, relevant zinc-iron transport protein exists Research in seed is also little.

In the expression pattern in embryo and endosperm, the present invention recognizes that ZmIRT1 mainly expresses in embryo by ZmIRT1 gene, So, the ZmIRT1 gene of the present invention is carried out overexpression in cereal crops seed can increase the long-pending of zinc iron content in seed Tired.

Therefore, the invention provides the ability of a kind of regulation and control plant absorption, transhipment or storage zinc-iron, release excessive zinc-iron To plant poisoning, the method for zinc iron content in the growth of regulation and control embryo or endosperm or maturation and increase cereal crops seed, including： By exercisable for ZmIRT1 gene of the present invention be connected with expression regulation element obtain recombinant plant expression vector；By recombinant plant Expression vector is transformed in plant, overexpression ZmIRT1 gene in plant；Particularly, by ZmIRT1 gene of the present invention in grain The content of zinc-iron in cereal crops seed when carrying out overexpression in food crop seed, can be effectively increased.

Invention further provides regulating and controlling the recombinant plant expression vector of transporter gene containing described zinc-iron and containing There is the host cell of this recombinant plant expression vector.

By the regulation and control of described zinc-iron, transporter gene is exercisable is connected with expression regulation element, and obtaining can be in plant Express the recombinant plant expression vector that this zinc-iron regulates and controls transporter gene." exercisable connection " refer to two or more elements it Between functional connection, the element of exercisable connection can be adjacent or non-adjacent.For example, this recombinant plant expression vector can So that by 5 ' end noncoding regions, the nucleotide shown in SEQ ID No.1 and 3 ' noncoding regions form, wherein, the 5 ' described non-volumes in end Code area can include promoter sequence, enhancer sequence or/and translation enhancement sequences；Described promoter can be that composition opens Mover, inducible promoter, tissue or organ specific promoters；3 ' described noncoding regions can comprise terminator sequence, MRNA cutting sequence etc..Suitable terminator sequence is retrieved from the Ti- plasmid of Agrobacterium tumefaciems, such as octopine synthase or rouge Fat alkali synzyme terminator.For example, in order that the zinc-iron regulation and control transporter gene of the present invention is carried out specifically in cereal crops seed Property expression, can by zinc-iron regulation and control transporter gene be connected to seed-specific expression promoter lower section build obtain restructuring plant Thing expression vector, after this recombinant plant expression vector transformation receptor plant, zinc-iron regulation and control transporter gene can be planted in receptor Carry out specific expressed in the seed of thing, reach the effect increasing seed zinc iron content or regulation and control embryonic development.

In addition, the nucleotide sequence shown in SEQ ID No.1 can be optimized to strengthen it for those skilled in the art Expression in plant.For example, the preference codon of target plant can be adopted to be optimized synthetic polyribonucleotidess to strengthen this Expression in target plant for the gene, these methods are known by those skilled in the art.

Additionally, this recombinant plant expression vector also can contain for selecting the selected marker of transformed cell.Select Property marker gene be used for select inverted cell or tissue.Described selected marker includes：Coding antibiotic resistance Gene and the gene etc. giving herbicides compounds resistance.Additionally, described marker gene also includes phenotypic markers, such as β- Tilactase and fluorescin etc..

Described " conversion " refers to will be hereditary to polynucleotide or polypeptide to the internal such mode of plant cell by channel genes It is transformed in plant.It is known by this area that described polynucleotide or polypeptide are incorporated into method in plant, including but do not limit In stable conversion method, transient transformation methods and virus-mediated methods etc..The polynucleotide constructs that " stable conversion " refers to be introduced into are integrated Simultaneously can be by its filial generation heredity to the genome of plant cell；" instantaneous conversion " refer to polynucleotide be introduced in plant but only Can transient expression or presence in plant.

Conversion scheme and the plant (monocotyledon that the scheme of described polynucleotide introduced plant is visually used for conversion Or dicotyledon) or the type of plant cell and change.The appropriate method that described polynucleotide are converted plant cell includes： Microinjection, electroporation, Agrobacterium-medialed transformation, direct gene transfer and high velocity ballistic bombardment etc..Implement specific In scheme, can be utilized multiple transient transformation methods that the ZmIRT1 gene of the present invention is supplied to plant.In other embodiments, originally The ZmIRT1 gene of invention can be by contacting and to be incorporated in plant with virus or viral nucleic acid plant, generally, such side Method is related to the ZmIRT1 gene construct of the present invention is introduced in viral DNA or RNA molecule.

Cell regeneration stable conversion plant (the McCormick et al.Plant of conversion can be made using conventional method Cell Reports.1986.5:81-84).The present invention can be used for converting any floristics, including but not limited to：Unifacial leaf is planted Thing or dicotyledon；Preferably, described target plant includes cereal crops, vegetable or fruit tree etc., and more preferably grain is made Thing, for example, it may be the cereal crops such as Semen Maydiss, Oryza sativa L., Fructus Hordei Vulgaris, Semen Tritici aestivi, Sorghum vulgare Pers., Semen sojae atricolor, Rhizoma Solani tuber osi.

The term definition that the present invention relates to

Unless otherwise defined, otherwise all technology used herein and scientific terminology all have with of the art Those of ordinary skill's generally understood identical implication.Although can use and described herein in the practice or test of the present invention It is similar to or equivalent any method, device and material, but presently describe method for optimizing, device and material.

Term " recombinant host cell strain " or " host cell " mean to comprise the cell of polynucleotide of the present invention, but regardless of making Inserted with which kind of method to produce recombinant host cell, for example directly known in picked-up, transduction, f pairing or art Other methods.Exogenous polynucleotide can remain the non-integrated vector of such as plasmid or can be integrated into host genome In.Host cell can be prokaryotic cell or eukaryotic cell, and host cell can be also unifacial leaf or dicotyledonous plant cells.

Term " nucleotide " means the deoxyribonucleotide of sub-thread or bifilar form, dezyribonucleoside, ribonucleotide Or ribonucleotide and its polymer.Except nonspecific restriction, otherwise described term is covered containing similar known to natural nucleotide The nucleic acid of thing, described analog have similar to reference nucleic acid binding characteristic and with the side similar to naturally-produced nucleotide Formula carries out metabolism.Unless in addition specific restriction, otherwise described term also means oligonucleotide analogs, and it includes PNA (peptide core Acid), DNA analog (thiophosphate, phosphamide acid esters etc.) used in antisense technology.Unless otherwise, otherwise Specific nucleic acid sequence also impliedly covers its conservative variant (including but not limited to degenerate codon replacement) modified and mutual Complementary series and the sequence clearly specified.Particularly, can be close by generation one of them or more than one selected (or all) The sequence that 3rd blended base of numeral and/or deoxyinosine residue replace replaces (Batzer etc. realizing degenerate codon People, Nucleic Acid Res.19:5081(1991)；Ohtsuka et al., J.Biol.Chem.260:2605-2608 (1985)；With Cassol et al., (1992)；Rossolini et al., Mol Cell.Probes 8:91-98(1994)).

Term " polypeptide ", " peptide " and " protein " used interchangeably herein is to mean the polymer of amino acid residue.That is, Description for polypeptide is equally applicable to describe peptide and description albumen, and vice versa.Described term is applied to naturally-produced ammonia Base acid polymer and one of or more than one amino acid residue are the amino acid polymer of non-naturally encoded amino acids.As Used herein, the amino acid chain of any length covered in described term, and it includes full-length proteins (i.e. antigen), wherein aminoacid Residue connects via covalent peptide bonds.

Brief description

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

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

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

Expression pattern during Semen Maydiss Embryo and endosperm development for Fig. 4 ZmIRT1 gene.

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

Fig. 6 pRTL2NGFP-ZmIRT1 recombinant vector enzyme action is identified.

Fig. 7 pRTL2NGFP-ZmIRT1 vector construction flow chart.

Subcellular Localization in Fig. 8 ZmIRT1 onion epidermis cell；GFP is pRTL2NGFP empty carrier positioning scenarios； ZmIRT1 is the Subcellular Localization situation of pRTL2NGFP-ZmIRT1.

Subcellular Localization in Fig. 9 ZmIRT1 arabidopsiss mesophyll protoplast；GFP positions feelings for pRTL2NGFP empty carrier Condition；ZmIRT1 is the Subcellular Localization situation of pRTL2NGFP-ZmIRT1.

Figure 10 pFL61-ZmIRT1 and pFL61-OsZIP5, pFL61-OsZIP8, pFL61-OsIRT1 positive connection restructuring Carrier enzyme action is identified；M is the Marker of 1Kb；1-4 be followed successively by pFL61-ZmIRT1, pFL61-OsZIP5, pFL61-OsZIP8, PFL61-OsIRT1 double digestion result.

Figure 11 pFL61-ZmIRT1 vector construction flow chart.

Figure 12 ZmIRT1 yeast complementation experiment result.

The schematic diagram of Figure 13 plant expression vector pBI121-ZmIRT1.

Figure 14 ZmIRT1 gene overexpression in arabidopsiss improves the experimental result of zinc or iron content in arabidopsiss； ZmIRT1 is the result turning ferrum and zinc-content determination in ZmIRT1 gene arabidopsiss seed；WT is in wild type Colombia seed Ferrum and the result of zinc-content determination.

Specific embodiment

To further describe the present invention with reference to specific embodiment, advantages of the present invention and feature will be with description and Apparent.But these embodiments are only exemplary, any restriction is not constituted to the scope of the present invention.People in the art Member should be understood that can be to enter to the details of technical solution of the present invention and form under without departing from the spirit and scope of the present invention Row modification or replacement, but these modifications and replacement each fall within protection scope of the present invention.

Experiment material

1.1 vegetable material

Corn inbred line X178 is carried by agricultural college of Agricultural University Of Hebei/country's corn improvement center Hebei branch center laboratory For Oryza sativa L. selfing line Japan is fine to be given by Beijing Normal University's school of life and health sciences.

1.2 bacterial strains and carrier

Escherichia coli (E.coli) bacterial strain Mach1-T1 and Agrobacterium (A.tumefacterium) bacterial strain EHA105, GV3101 is preserved by this laboratory.PGEM-Teasy carrier is purchased from Promega company.Yeast expression carrier pFL61 (schematic diagram See Fig. 1), 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) given by Agricultural University Of Nanjing Zhang Hongsheng professor's friendship.

Embodiment 1 Semen Maydiss zinc-iron regulates and controls the clone of transporter ZmIRT1 gene

1st, the process of vegetable material

First Vermiculitum is impregnated with Hoagland nutritional solution, corn inbred line X178 seed point is sowed in seedlings nursing plate, above Cover the dry Vermiculitum of last layer, culture in greenhouse (16h illumination/8h is dark, 26 DEG C), 12 days seedling length to 2 leaves wholeheartedly when move into In standard Hoagland nutritional solution, growth wholeheartedly (changes one time of nutrition liquid in every 3 days) to 3 leaves in 6 days, and 3 leaves corn seedling wholeheartedly is in mark Process 0 under conditions of quasi- nutritional solution and not zincification, ferrum, copper, manganese and high zinc, ferrum, 6,12,24,48, after 96h, collect seedling respectively Overground part and root, liquid nitrogen flash freezer preserves for Total RNAs extraction after -80 DEG C.

2nd, the extraction of Semen Maydiss total serum IgE

Semen Maydiss total serum IgE is extracted using Trizol method.

3rd, the synthesis of cDNA

(1), remove DNA, by following preparation reaction systems：Total serum IgE (1 μ g/ μ L) 1.0 μ L, DNAse I (10U/ μ L) 1.0 μ L, 10 × DNAse I buffer 1.0 μ L, DEPC H₂O 7.0 μ L, amounts to 10.0 μ L；37 DEG C of 30min, add 1 μ L 25mM EDTA, 65 DEG C of 5min terminating reactions.

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

(3), totally 12 μ L above, adds following components and obtains 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 mix 2.0 μ L, amount to：20.0μL；42 DEG C of 60min, 70 DEG C of 5min, terminating reaction.

4th, the clone of genes of interest

(1), the ORF frame according to genes of interest designs primer：

ZmIRT1F 5'-GCGGCCGCTCTAGAATGTCTTGGCGGCGAAACC-3'NotI,XbaI

ZmIRT1R 5'-GCGGCCGCTACGTATCA CGCCCACTTGGCCATGATG-3'NotI,SnaBI

With the cDNA of above-mentioned steps 3 as template, from ExTaq enzyme, 2 × GCI buffer enters performing PCR amplification, PCR program For：95 DEG C of denaturations 4min；94 DEG C of degeneration 1min, 60 DEG C of annealing 1min, 72 DEG C of extension 1min, 33 circulations；72 DEG C of extensions 10min；

(2), the fragment obtaining clone is cloned in pGEM-T carrier, converts Mach1-T1 bacterial strain；

(3), obtain positive recombiant plasmid through enzyme action identification, be named as pGEM-ZmIRT1, sequencing is correctly cloned. The unnamed gene cloned is ZmIRT1, the cDNA sequence of this gene is shown in SEQ ID No.1, the aminoacid sequence derived It is classified as shown in SEQ ID No.2, its whole genome sequence is shown in SEQ ID No.3.

Expression pattern in seedling, embryo and endosperm for embodiment 2 ZmIRT1

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

CDNA 2.0 μ L, ExTaq 0.1 μ L, 2 × GCI buffer 10.0 μ L, 10mM dNTP mix 0.8 μ L, upstream is drawn Thing RTZmIRT1F (10 μM/μ L) 1.0 μ L, downstream primer RTZmIRT1R (10 μM/μ L) 1.0 μ L, ddH₂O 5.1 μ L, amounts to 20.0μL；

RTZmIRT1F 5'-CACCACCTTCGTCGCCAT-3'

RTZmIRT1R 5'-TGTTGCCACCCTTCCTCC-3'

PCR reaction condition：94 DEG C of denaturations 4min；30 circulations, each 94 DEG C of degeneration of circulation 45 seconds, 60 DEG C of annealing 1min, 72 DEG C of extension 1min；Finally re-extend 72 DEG C of 10min, 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：The cDNA of reverse transcription in embodiment 1 step 3 dilutes 20 times as Real-time The template of PCR reaction, Actin is internal reference, and reaction system is as follows：

CDNA 5.0 μ L, SYBR Green I 10.0 μ L, Rox 0.4 μ L, forward primer ZmActin1F (10 μM/μ L) 0.4 μ L, downstream primer ZmActin1R (10 μM/μ L) 0.4 μ L, ddH₂O3.8 μ L, amounts to 10.0 μ L；

ZmActin1F 5'-ATGTTTCCTGGGATTGCCGAT-3'

ZmActin1R 5'-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 the conditions of normal nutrition, ZmIRT1 portion and underground on the ground There is expression in portion, expression higher (Fig. 2) in root, and under the conditions of zinc deficiency, ZmIRT1 is in 96h overground part up-regulated；ZmIRT1 Under the conditions of high zinc, the expression of overground part gradually rises, and reaches highest during 96h, however, the expression of underground part reduces (figure 3).These results indicate that ZmIRT1 is more sensitive to zinc concentration in seedling period.

Under the conditions of iron deficiency, ZmIRT1 overground part up-regulated in 96h, underground part expression significantly increases, high ferro condition Under, whether on the ground or the expression of underground is all to reduce (Fig. 2), these results show that ZmIRT1 is dense to ferrum to ZmIRT1 Degree is more sensitive.

Expression analysis in the embryo and endosperm of different number of days after pollination find, ZmIRT1 is mainly in the later stage table of embryonic development The amount of reaching substantially raises (Fig. 4), illustrates that ZmIRT1 plays a role in the growth course of embryo.

The bioinformatic analysis of embodiment 3 ZmIRT1

Bioinformatic analysis show, zinc-iron regulation and control transporter ZmIRT1 is positioned on the 1st chromosome of Semen Maydiss, and zinc-iron is adjusted Control transporter ZmIRT1 is made up of 381 aminoacid, containing 8 membrane spaning domains, has a richness between the 3rd and the 4th transmembrane region Variable region containing histidine, may be relevant with the binding transport of metal ion.Phylogenetic analysis show, ZmIRT1 and AtIRT1, AtIRT2, OsIRT1, OsIRT2 evolutionary relationship is relatively nearly (Fig. 5) thus it is speculated that ZmIRT1 is probably iron transporters.

The Subcellular Localization of embodiment 4 ZmIRT1

1st, the structure of fusion expression vector

According to the primers of ZmIRT1 gene, primer sequence is as follows：

ZmIRT1GF 5'-CTCGAGATGTCTTGGCGGCGAAACC-3'XhoI

ZmIRT1GR 5'-TCTAGACGCCCACTTGGCCATGATG-3'XbaI

Add suitable restriction enzyme site, and gene 3 ' end removes termination codon, is connected to pGEM- during with clone gene The correct plasmid that is sequenced in carrier T is template, enters performing PCR amplification from ExTaq enzyme and 2 × GCI buffer, and PCR program is： 95 DEG C of denaturations 4min；94 DEG C of degeneration 1min, 60 DEG C of annealing 1min, 72 DEG C of extension 1min, 33 circulations；72 DEG C of extension 10min. Amplified fragments reclaim rear clone in pGEM-T carrier through 1% agarose gel electrophoresiies, convert coli strain Mach1-T1, Obtain positive colony, upgrading grain, enzyme action and sequence verification through LB culture medium (IPTG, X-gal, Amp)；To connect during clone gene The correct plasmid that is sequenced on pGEM-T carrier is template, enters performing PCR amplification, amplification from ExTaq enzyme and 2 × GCI buffer Fragment reclaims rear clone in pGEM-T carrier through 1% agarose gel electrophoresiies, after the correct plasmid enzyme restriction that is sequenced, by purpose piece Section is building up on pRTL2NGFP carrier, is named as pRTL2NGFP-ZmIRT1, and Fig. 6 is enzyme action qualification figure, and Fig. 7 is to build flow process Figure.

2nd, with corresponding enzyme action pRTL2NGFP carrier and the gene after different enzyme action, through T₄DNA ligase connects, and turns Change Mach1-T1 bacterial strain, carry plasmid enzyme restriction evaluation and screening and go out correct recombinant big upgrading grain for via Particle Bombardment Transformation onion epidermis.

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

4th, carry out onion epidermis conversion with particle gun

Understand that zinc-iron regulation and control transporter ZmIRT1 is positioned at intercellular membrane (Fig. 8) from positioning result.For further determining that The plasma membrane of cell and endomembrane system, carry out the conversion of arabidopsiss mesophyll protoplast from ER marker, and result proves that zinc-iron is adjusted Control transporter ZmIRT1 is positioned at (Fig. 9) in cytoplasma membrane and endoplasmic reticulum.

Embodiment five yeast complementation experiment

1st, the structure of Yeast expression carrier

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

ZmIRT1YF 5'-GCGGCCGCTCTAGAATGTCTTGGCGGCGAAACC-3'NotI,XbaI

ZmIRT1YR 5'-GCGGCCGCTACGTATCACGCCCACTTGGCCATGATG 3'NotI,SnaBI

It is connected on pGEM-T carrier the correct plasmid that is sequenced as template, from ExTaq and 2 × GCI during with clone gene Buffer enters performing PCR amplification, and PCR program is：95 DEG C of denaturations 4min；94 DEG C of degeneration 1min, 60 DEG C of annealing 1min, 72 DEG C of extensions 1min, 33 circulations；72 DEG C of extension 10min.Amplified fragments reclaim rear clone to pGEM-T carrier through 1% agarose gel electrophoresiies In, convert coli strain Mach1-T1, obtain positive colony, upgrading grain, enzyme through LB culture medium (IPTG, X-gal, Amp) Cut and sequence verification, be sequenced correct plasmid after corresponding enzyme action, purpose fragment be building up on pFL61 carrier, be named as PFL61-ZmIRT1 and pFL61-OsZIP5, pFL61-OsZIP8 and pFL61-OsIRT1, Figure 10 is enzyme action qualification figure, Tu11Wei Build flow chart.

With the ZmIRT1 fragment after NotI enzyme action pFL61 carrier and enzyme action through T₄DNA ligase connects, and converts Mach1-T1 Bacterial strain, carries plasmid enzyme restriction evaluation and screening and goes out correct recombinant big upgrading grain for transformed saccharomyces cerevisiae.

2nd, electroporated method transformed yeast

(1), from YPD flat board the single bacterium colony of picking zrt1zrt2ZHY3, fet3fet4DEY1453 and DY1455 in 20mL YPD fluid medium in, 28 DEG C of shaking table culture about 24h；

(2) bacterium solution, drawing above 2% volume is transferred to continuation expanding propagation about 4-5h in the YPD culture medium of 100mL, treats bacterium Liquid OD₆₀₀For competence can be prepared during 1.2-1.5；

(3), bacterium solution is collected in the centrifuge tube of 50mL, 4 DEG C, 5,000rpm, it is centrifuged 5min, outwell supernatant；

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

(5) deionized water of 1/2 volume, resuspended thalline on ice, are added, 4 DEG C, 5,000rpm, 5min are centrifuged, and outwell Clearly；

(6) the 1M sorbitol solution of 10mL, resuspended thalline on ice, are added, 4 DEG C, 5,000rpm, 5min are centrifuged, and outwell Clearly；

(7), add the sorbitol solution of 450-600 μ L, gently inhaled with the pipette tips decaptitating, resuspended thalline；

(8), it is defined according to the competence adding about 100 μ L in the centrifuge tube of each 1.5mL, subpackage；

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

(10), electroporated, add about 800 μ L, the sorbitol solution of the pre-cooling of 1M, resuspended thalline immediately；

(11), from electric shock cup, suction out thalline, be coated with SD/Ura- flat board；

(12), on SD flat board, 28 DEG C of cultures can grow macroscopic bacterial plaque in about 6 days.

3rd, the identification of yeast-positive clone

(1), take 1.5mL yeast culture, 9,000rpm centrifugations 30 seconds, inhale as far as possible and abandon supernatant, collect yeast cells；

(2), add 600 μ L Sorbitol buffer, softly blow and beat abundant re-suspended cell, add the Lyticase of 80U, Fully reverse mixing, 37 DEG C of incubation 30min peptic cell walls, centre is reverse for several times；

(3), 13,000rpm centrifugation 1min, inhales as far as possible and abandons supernatant, adds the resuspended bacterial sediment of 250 μ L solution YP1, whirlpool Rotation concussion suspends to thorough；

(4), add 250 μ L YP2 solution, gently overturn, so that thalline is fully cracked, room temperature places 4min；

(5), add 350 μ L YP3 solution, gently overturn, when fully mixing, white flock precipitate occurs, quiet on ice Put 3-5min, 13,000rpm centrifugation 5min, careful Aspirate supernatant.

(6), by previous step gained supernatant add adsorption column AC in (adsorption column is put in collecting pipe), 12,000rpm from The heart 30-60 second, outwell the waste liquid in collecting pipe；

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

(8), add 500 μ L rinsing liquid WB (plus dehydrated alcohol), 12,000rpm centrifugation 30-60 seconds, abandon waste liquid；

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

(10), adsorption column AC is put back in sky collecting pipe, 13,000rpm centrifugation 2min, remove rinsing liquid；

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

(12), with 1 μ L DNA of extracting as template, the two ends primer of gene is pcr amplification primer thing, enters performing PCR amplification and tests Card genes of interest, verifies correct bacterium solution, adds 25% glycerol to preserve in -80 DEG C.

4th, yeast complementation experiment

By pFL61, pFL61-ZmIRT1, pFL61-OsZIP5, pFL61-OsZIP8, pFL61-OsIRT1 convert ferment respectively Female mutant zrt1zrt2ZHY3 and fet3fet4DEY1453, pFL61 are negative control, and OsZIP5, OsZIP8 are zinc transporter Positive control (Lee S, Kim SA, Lee J, et al.Zinc deficiency-inducible OsZIP8encodes a 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 experimental botany 2007,58(11):2909-2915.), OsIRT1 is positive control (Lee S, the An G.Over- of iron transporters expression of OsIRT1leads to increased iron and zinc accumulations in rice.Plant,cell&environment 2009,32(4):408-416.), pFL61 conversion wild-type strain DY1455 makees For another positive control, after conversion, it is accredited as the yeast of the positive, cultivates in SD fluid medium, yeast liquid dilutes respectively 4 concentration (OD₆₀₀=1,0.1,0.01,0.001) 5 μ L points, are then taken in the culture medium of low zinc, low ferrum and normal SD, low zinc (SD culture medium adds 0.4mM EDTA, 0.4mM EDTA and 250 μM of ZnSO to culture medium₄, 0.4mM EDTA and 300 μM ZnSO₄), (SD culture medium adds 50mM MES, 50mM MES and 50 μM of FeCl to low ferrum culture medium₃, 50mM MES and 100 μM FeCl₃) yeast complementation reference Lin, test (Lin YF, the Liang HM, Yang SY, et al.Arabidopsis of Y.F IRT3is a zinc-regulated and plasma membrane localized zinc/iron transporter.The New phytologist 2009,182(2):392-404.) method is carried out, 28 DEG C of cultures, sees within 6 days Examine result of the test.

Yeast complementation experiment result shows, under the conditions of low zinc, added with 250 μM of ZnSO₄Culture medium in can substantially observe Better than empty carrier pFL61 growing way to DY-pFL61 (wild type), Z-ZmIRT1, Z-OsZIP5, Z-OsZIP8, Z-OsIRT1；And And, ZmIRT1 than it has been reported that rice Os ZIP growing way good.Under the conditions of low ferrum, D-ZmIRT1 ratio it has been reported that OsIRT1 Transport activity be also eager to excel (Figure 12), though ZmIRT1 in low zinc or all showing extremely strong zinc-iron under the conditions of low ferrum transports Activity, illustrates that ZmIRT1 plays a major role to absorbing and transporting zinc-iron during corn growth.

Experimental example 1 ZmIRT1 gene overexpression in arabidopsiss improves the experiment of ferrum and Zn content in arabidopsiss seed

The plant expression vector pBI121 that ZmIRT1 gene is controlled with the composing type 35S promoter structure that is connected obtains ZmIRT1 genetically modified plants expression vector (Figure 13)；The recombinant plant expression vector of structure is transformed in arabidopsiss, identification Obtain the positive and turn ZmIRT1 gene arabidopsiss；The positive is turned ZmIRT1 gene arabidopsiss carry in identical with wild type Colombia Cultivate under the conditions of training, harvest and turn ZmIRT1 gene arabidopsiss seed and wild type seeds, measure respectively and turn ZmIRT1 gene plan south The content of ferrum and zinc in canola seed and wild type seeds；Weigh a certain amount of vegetable material through micro-wave digestion, constant volume, use ICP-MS Method carries out the mensure of zinc iron content.Every batch of mensure 200mg seed, measures three batches, takes the meansigma methodss of three batch datas.Measurement result See Figure 14.From the result of Figure 14, in arabidopsiss, overexpression ZmIRT1 gene can effectively improve ferrum and zinc in seed and contain The content of amount, wherein ferrum improves especially pronounced.

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.