CN104212816B - Maize zinc-iron regulation transporter ZmZIPs gene and application thereof - Google Patents
Maize zinc-iron regulation transporter ZmZIPs gene and application thereof Download PDFInfo
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- CN104212816B CN104212816B CN201410240908.XA CN201410240908A CN104212816B CN 104212816 B CN104212816 B CN 104212816B CN 201410240908 A CN201410240908 A CN 201410240908A CN 104212816 B CN104212816 B CN 104212816B
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- 239000002689 soil Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 230000009469 supplementation Effects 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- RYYWUUFWQRZTIU-UHFFFAOYSA-K thiophosphate Chemical compound [O-]P([O-])([O-])=S RYYWUUFWQRZTIU-UHFFFAOYSA-K 0.000 description 1
- 239000011573 trace mineral Substances 0.000 description 1
- 235000013619 trace mineral Nutrition 0.000 description 1
- 238000010361 transduction Methods 0.000 description 1
- 230000026683 transduction Effects 0.000 description 1
- 230000010474 transient expression Effects 0.000 description 1
- 229910052723 transition metal Inorganic materials 0.000 description 1
- 150000003624 transition metals Chemical class 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
- 229910001868 water Inorganic materials 0.000 description 1
- 210000005253 yeast cell Anatomy 0.000 description 1
- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 description 1
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Abstract
The invention discloses a maize zinc-iron regulation transporter ZmZIPs gene and application thereof. The invention separates 5 zinc-iron control transporters ZmZIPs genes from corn, and the cDNA sequences are respectively selected from the nucleotide sequences shown in SEQ ID No.1, SEQ ID No.3, SEQ ID No.5, SEQ ID No.7 or SEQ ID No. 9. Subcellular localization indicates that zinc-iron regulatory transporters encoded by the genes are localized in plasma membranes and endoplasmic reticulum of cells. Yeast complementary experiments show that the 5 ZmZIPs genes separated by the invention have zinc-iron transport function. The ZmZIPs gene has important application prospect in the aspects of regulating and controlling the capacity of plants to absorb, transport and store zinc and iron, promoting the development and maturation of embryos and endosperms, increasing the content of zinc and iron in crop seeds and the like.
Description
Technical field
The present invention relates to from plant detached metal-ions transportation body gene, detached and zinc more particularly, to from corn
The relevant regulation and control transporter ZmZIPs gene of the absorption of iron, transhipment or storage, the invention further relates to regulation and control transporter
ZmZIPs gene is regulating and controlling plant absorption, transhipment or storage zinc or iron ability, the growth of promotion embryo and endosperm or ripe and increasing
Plus 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 iron are trace element, important role in the growth and development process of plant necessary to organism
(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 chemistry2003,278(48):47644-47653.).Zinc
It is structure confactor (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 physiology2007,143(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 chemistry2005,280
(1):787-795.).The appropriate content increasing zinc in plant body can improve crop yield, and the shortage of zinc can lead to chlorophyll,
Lipid, albumen, the Oxidative demage of plasma membrane, in plant body, zinc ion builds up and can produce murder by poisoning to plant.
Iron plays a significant role in the catalytic reaction process of cellular respiration, photosynthesis and metalloprotein, is important
Electron transit mediator, therefore, ferro 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 vie1999,322(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 iron 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 biology2006,9
(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 Acta2000,1465(1-2):190-198.).
Identify ZIP in the plants such as arabidopsis, paddy rice, puncture vine, clover, soybean, wild type emmer wheat, grape at present
Gene is simultaneously studied to its function.Find 16 ZIP family genes in arabidopsis, 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 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 in root, fixed
Position is in vesica 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 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 mutually zinc supplementation, iron transfer double-mutant, overexpression AtIRT3 can make zinc portion on the ground
And iron accumulates (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
phytologist2009,182(2):392-404.).Expression analysis show, AtZIP1, AtZIP5, AtZIP9, AtZIP12 and
AtIRT3 be subject to lack zinc induce, thus speculate, these genes under the conditions of scarce zinc may strengthen zinc absorbability (Kramer U,
Talke IN,Hanikenne M.Transition metal transport.FEBS Lett2007,581(12):2263-
2272.).
Corn (Zea mays) is the important grain of China, feed and industrial crops, increases zinc, iron 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,
Known many transport proteins 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, iron etc. two
The absorption of valence metal ion, transport and storage play an important role, and on arabidopsis, paddy rice, barley, soybean, 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 corn is less.Understand Zn2+、Fe2+Absorption in corn, means of transportation, point
Cloth rule and regulation mechanism, it will help improve corn growing in zinc, sideropenic environment, for disclosing jade further
In rice, the mechanism of action of ZIP family gene lays the foundation, and provides candidate gene for corn zinc, iron 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 corn (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, promote radicle and embryonic development and embryo maturation or increase zinc in cereal crops seed
Iron content.
The above-mentioned purpose of the present invention is achieved through the following technical solutions：
From corn (Zea mays), detached zinc-iron regulates and controls transporter ZmZIP1 gene, and its cDNA sequence is (a), (b)
Or shown in (c):
Nucleotide sequence shown in (a), SEQ ID No.1；
(b), the nucleotide sequence of amino acid shown in coding SEQ ID No.2；
C nucleosides that () and the complementary series of nucleotides shown in SEQ ID No.1 can be hybridized in stringent hybridisation conditions
Acid, the protein coded by this nucleotides has the function that zinc-iron regulates and controls transporter.
From corn (Zea mays), detached zinc-iron regulates and controls transporter ZmZIP2 gene, and its cDNA sequence is (a), (b)
Or shown in (c):
Nucleotide sequence shown in (a), SEQ ID No.3；
(b), the nucleotide sequence of amino acid shown in coding SEQ ID No.4；
C nucleosides that () and the complementary series of nucleotides shown in SEQ ID No.3 can be hybridized in stringent hybridisation conditions
Acid, the protein coded by this nucleotides has the function that zinc-iron regulates and controls transporter.
From corn (Zea mays), detached zinc-iron regulates and controls transporter ZmZIP3 gene, and its cDNA sequence is (a), (b)
Or shown in (c):
Nucleotide sequence shown in (a), SEQ ID No.5；
(b), the nucleotide sequence of amino acid shown in coding SEQ ID No.6；
C nucleosides that () and the complementary series of nucleotides shown in SEQ ID No.5 can be hybridized in stringent hybridisation conditions
Acid, the protein coded by this nucleotides has the function that zinc-iron regulates and controls transporter.
From corn (Zea mays), detached zinc-iron regulates and controls transporter ZmZIP7 gene, and its cDNA sequence is (a), (b)
Or shown in (c):
Nucleotide sequence shown in (a), SEQ ID No.7；
(b), the nucleotide sequence of amino acid shown in coding SEQ ID No.8；
C nucleosides that () and the complementary series of nucleotides shown in SEQ ID No.7 can be hybridized in stringent hybridisation conditions
Acid, the protein coded by this nucleotides has the function that zinc-iron regulates and controls transporter.
From corn (Zea mays), detached zinc-iron regulates and controls transporter ZmZIP8 gene, and its cDNA sequence is (a), (b)
Or shown in (c):
Nucleotide sequence shown in (a), SEQ ID No.9；
(b), the nucleotide sequence of amino acid shown in coding SEQ ID No.10；
C nucleosides that () and the complementary series of nucleotides shown in SEQ ID No.9 can be hybridized in stringent hybridisation conditions
Acid, the protein coded by this nucleotides has the function that zinc-iron regulates and controls transporter.
Described " stringent hybridisation conditions " mean the condition of known LIS 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 1.0M in pH7.0 to 8.3 times salinity
Na ion concentration, typically about 0.01 arrives 1.0M Na ion concentration (or other salt), and temperature for short probe (include (but
Be not limited to) 10 to 50 nucleotides) for be at least about 30 DEG C, and (50 cores are including but not limited to more than for long probe
Thuja acid) for be at least about 60 DEG C.High stringency conditions also can be realized by adding the destabilizing agent of such as formamide.For selection
Property or specific hybrid for, positive signal can be at least twice background hybridization, optionally for 10 times of background hybridizations.Exemplary tight
Careful hybridization conditions can be as follows：50% formamide, 5 × SSC and 1%SDS, cultivate at 42 DEG C；Or 5 × SSC, 1%SDS, 65
Cultivate at DEG C, washing and washing in 0.1%SDS at 65 DEG C in 0.2 × SSC.Described washing can carry out 5,15,30,60,
120min or longer time.
Preferably, the present invention separated from corn zinc-iron regulation and control transporter ZmZIP1, ZmZIP2, ZmZIP3,
The cDNA sequence of ZmZIP7 or ZmZIP8 gene is respectively SEQ ID No.1, SEQ ID No.3, SEQ ID No.5, SEQ ID
Nucleotide sequence shown in No.7 or SEQ ID No.9.
The two of the object of the invention be provide by above-mentioned zinc-iron regulation and control transporter ZmZIPs gene (ZmZIP1, ZmZIP2,
ZmZIP3, ZmZIP7 or ZmZIP8) coded by the protein that can absorb, transport or store zinc-iron；
The two of the object of the invention are achieved through the following technical solutions：
The zinc-iron regulation and control transporter of ZmZIPs coded by said gene, its amino acid sequence is shown in (a) or (b):
Shown in (a), SEQ ID No.2, SEQ ID No.4, SEQ ID No.6, SEQ ID No.8 or SEQ ID No.10
Amino acid sequence；
(b), by SEQ ID No.2, SEQ ID No.4, SEQ ID No.6, SEQ ID No.8 or SEQ ID No.10 institute
The amino acid sequence showing by the replacement of one or more amino acid residues, disappearance or/and insertion derivative obtain still have
The protein variant of zinc-iron regulation and control transhipment body function.
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 amino acid 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 methods of operating 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 mutagenesis or change polynucleotides method be this area known by.Wherein, conservative replacement is will be residual for a kind of amino acid
Base is substituted for another kind of amino acid 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 polynucleotides, variant
Comprise disappearance, insertion or/and the replacement of the one or more nucleotides of one or more site in native polynucleotide.For many
Nucleotides, conservative variant includes not changing those variants of the amino acid 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 polynucleotides in source, for example, using the many nucleosides still encoding the amino acid shown in SEQ ID No.2 obtained by direct mutagenesis
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 genetically modified plants etc..
Knowable to Subcellular Localization result, the detached 5 ZmZIPs genes of the present invention (ZmZIP1, ZmZIP2, ZmZIP3,
ZmZIP7 or ZmZIP8 gene) coded by albumen be positioned on plasma membrane and intercellular membrane.For further determining that intercellular membrane
Concrete positioning, the present invention carries out turning of arabidopsis mesophyll protoplast from ER marker and 5 ZmZIPs gene common locations
Change, result proves that this 5 ZmZIPs genes are both positioned in cytoplasma membrane and endoplasmic reticulum.On the basis of Subcellular Localization, lead to
Cross real-time RT-PCR expression analysis to find, under the conditions of normal nutrition, 5 ZmZIPs gene main portion on the ground expression,
Wherein, under the conditions of scarce zinc, in 96h overground part up-regulated, overground part and underground part expression be all in 6h for ZmZIP3 for ZmZIP8
Raise；Expression in high zink rod part ZmZIP7 and ZmZIP8 portion on the ground is gradually lowered, and ZmZIP3 is in underground part
Expression significantly reduce.These results indicate that ZmZIP3, ZmZIP7 and ZmZIP8 compared to zinc concentration in seedling period
Sensitive.Under the conditions of iron deficiency, ZmZIP7 and ZmZIP8 is gradually increased with the expression of underground on the ground, illustrate ZmZIP7 and
ZmZIP8 is more sensitive to the concentration of iron.Under the conditions of scarce copper, manganese deficiency, the expression of 5 ZmZIPs genes does not all significantly become
Change.
Yeast complementation experiment shows, and though 5 separated ZmZIPs of the present invention low zinc or under the conditions of low iron all
Show different degrees of transhipment zinc or iron activity, illustrate that 5 separated ZmZIPs of the present invention are respectively provided with the work(of transhipment zinc-iron
Energy.
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 barley, overexpression AtZIP1 gene can increase zinc and iron 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 biology2004,54(3):373-
385.), equally, overexpression OsIRT1 gene, in paddy rice, the content of zinc and iron 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&environment2009,32(4):408-416.).However, in paddy rice
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 cells2010,
29(6):551-558；Lee S,Jeong HJ,Kim SA,et al.OsZIP5is a plasma membrane zinc
transporter in rice.Plant molecular biology2010,73(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.) it is not reaching to increase the purpose of Zn content, therefore, these genes in seed
Overexpression is unfavorable to the enrichment of zinc in rice grain.These results indicate that dystopy overexpression for zinc-iron accumulation with point
Cloth may play a role.However, the research in seed is also little about zinc-iron transport protein.
Therefore, the invention provides a kind of regulation and control plant absorption, transhipment or storage zinc-iron ability method, including：Will
ZmZIPs gene of the present invention is exercisable to be connected with expression regulation element and obtains recombinant plant expression vector；By recombinant plant table
Reach carrier to be transformed in plant, overexpression ZmZIPs gene in plant, being capable of Effective Regulation or improve target plant to zinc
The ability of the absorption of iron, transhipment or storage.
The invention provides a kind of release the method that excessive zinc-iron is poisoned to plant, the method includes：By the present invention
ZmZIPs gene is exercisable to be connected with expression regulation element and obtains recombinant plant expression vector；By recombinant plant expression vector
It is transformed in plant, overexpression ZmZIPs gene in plant.
Present invention also offers a kind of regulation and control or the method promoting vegetable seeds embryonic development, the method includes：By the present invention
ZmZIPs gene is exercisable to be connected with expression regulation element and obtains recombinant plant expression vector；By recombinant plant expression vector
It is transformed in plant, overexpression ZmZIPs gene in plant；Wherein, in described expression regulation element, promoter is preferably
The promoter of seed-specific expression.
Invention further provides a kind of regulation and control or promote the ripe method of seed embryo, the method includes：By the present invention
ZmZIPs gene is exercisable to be connected with expression regulation element and obtains recombinant plant expression vector；By recombinant plant expression vector
It is transformed in plant, overexpression ZmZIPs gene in plant；Wherein, the promoter in described expression regulation element is preferred
Promoter for seed-specific expression.
Invention further provides regulate and control the recombinant plant expression vector of transporter ZmZIPs gene containing described zinc-iron
And the host cell containing this recombinant plant expression vector.
By zinc-iron of the present invention regulation and control, transporter ZmZIPs gene is exercisable is connected with expression regulation element, obtains
The recombinant plant expression vector that this zinc-iron regulates and controls transporter gene can be expressed in plant." exercisable connection " refer to two or
Functional connection between more elements, the element of exercisable connection can be adjacent or non-adjacent.For example, this restructuring is planted
Thing expression vector can be by 5 ' end noncoding regions, SEQ ID No.1 (or SEQ ID No.3, SEQ ID No.5, SEQ ID
Any sequence in No.7 or SEQ ID No.9) shown in nucleotides and 3 ' noncoding regions composition, wherein, described 5 ' ends are non-
Code area can include promoter sequence, enhancer sequence or/and translation and strengthen sequence；Described promoter can be composition
Promoter, inducible promoter, tissue or organ specific promoters；3 ' described noncoding regions can comprise terminator sequence
Row, mRNA cutting sequence etc..Suitable terminator sequence is retrieved from the Ti- plasmid of Agrobacterium tumefaciems, such as octopine synthase
Or nopaline synthase termination area.For example, in order that the zinc-iron regulation and control transporter gene of the present invention is carried out in cereal crops seed
Specific expressed, the lower section that zinc-iron regulation and control transporter gene can be connected to seed-specific expression promoter builds and obtains weight
Group plant expression vector, after this recombinant plant expression vector transformation receptor plant, zinc-iron regulation and control transporter gene can be subject to
Carry out specific expressed in the seed of body plant, reach promotion radicle and embryonic development, promote embryo ripe or increase seed zinc-iron
Content or the effect of regulation and control embryonic development.
In addition, the nucleotide sequence of ZmZIPs can be optimized to strengthen it in plant for those skilled in the art
Expression.For example.The preference codon that target plant can be adopted is optimized synthetic polyribonucleotides to strengthen this gene in target
Expression in plant, 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-
Galactosidase and fluorescin etc..
Described " conversion " refers to will be hereditary to polynucleotides 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 polynucleotides 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 polynucleotides be introduced in plant but only
Can transient expression or presence in plant.
Conversion scheme and the plant (monocotyledon that the scheme of described polynucleotides introduced plant is visually used for conversion
Or dicotyledon) or the type of plant cell and change.The appropriate method that described polynucleotides 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 ZmZIPs gene of the present invention is supplied to plant.In other embodiments, originally
The ZmZIPs 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 ZmZIPs gene construct of the present invention is introduced in viral DNA or RNA molecule.
Cytothesis 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, vegetables or fruit tree etc., and more preferably grain is made
Thing, for example, it may be the cereal crops such as corn, paddy rice, Resistance In Wheat And Barley, Chinese sorghum, soybean, potato.
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 polynucleotides 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 or eukaryotic, and host cell can be also unifacial leaf or dicotyledonous plant cells.
Term " nucleotides " 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 nucleotides
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 realizing degenerate codon.
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 amino acid
Residue connects via covalent peptide bonds.
Brief description
The schematic diagram of Fig. 1 Yeast expression carrier pFL61.
The expression pattern of ZmZIPs under Fig. 2 standard Hoagland culture medium condition；S (shoot), R (root).
Expression pattern under various treatment conditions for Fig. 3 ZmZIPs gene.
Expression pattern during corn Embryo and endosperm development for Fig. 4 ZmZIPs gene.
Fig. 5 pRTL2NGFP-ZmZIPs recombinant vector digestion is identified；M is the Marker of 1Kb；1-5 is respectively
The result of pRTL2NGFP-ZmZIP1, ZmZIP2, ZmZIP3, ZmZIP7, ZmZIP8 double digestion
Subcellular Localization in Fig. 6 ZmZIPs onion epidermis cell；GFP is pRTL2NGFP empty carrier positioning scenarios；
ZmZIP1, ZmZIP2, ZmZIP3, ZmZIP7, ZmZIP8 are the Subcellular Localization situation of pRTL2NGFP-ZmZIP1,2,3,7,8.
Subcellular Localization in Fig. 7 ZmZIPs arabidopsis mesophyll protoplast；GFP positions feelings for pRTL2NGFP empty carrier
Condition；GFP is pRTL2NGFP empty carrier positioning scenarios；ZmZIP1, ZmZIP2, ZmZIP3, ZmZIP7, ZmZIP8 are respectively
The Subcellular Localization situation of pRTL2NGFP-ZmZIP1,2,3,7,8.
Fig. 8 pFL61-ZmZIPs and pFL61-OsZIP5, pFL61-OsZIP8, pFL61-OsIRT1 positive connection restructuring
Carrier digestion is identified；M is the Marker of 1Kb；1-8 be followed successively by pFL61-ZmZIP1, pFL61-ZmZIP2, pFL61-ZmZIP3,
PFL61-ZmZIP7, pFL61-ZmZIP8, pFL61-OsZIP5, pFL61-OsZIP8, pFL61-OsIRT1 double digestion result.
Fig. 9 ZmZIPs yeast complementation experiment result.
The plant expression vector schematic diagram of Figure 10 ZmZIP1, ZmZIP2, ZmZIP3, ZmZIP7 and ZmZIP8.
Figure 11 ZmZIPs gene overexpression in arabidopsis improves the experimental result of Zn content in arabidopsis；ZmZIP2 is
Turn the experimental result that ZmZIP2 gene overexpression in arabidopsis improves iron or Zn content in arabidopsis；ZmZIP3 is to turn ZmZIP3
Gene overexpression in arabidopsis improves the experimental result of iron or Zn content in arabidopsis；ZmZIP7 is to turn ZmZIP7 gene intending
In southern mustard, overexpression improves the experimental result of iron or Zn content in arabidopsis；ZmZIP8 is to turn ZmZIP8 gene mistake in arabidopsis
Expression improves the experimental result of iron or Zn content in arabidopsis；WT is iron and zinc-content determination in wild type Colombia seed
Result.
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 paddy rice self-mating system 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/can1 his3/his3 leu2/leu2 trp1/
trp1 ura3/ura3fet3-2::HIS3/fet3-2::HIS3fet4-1::LEU2/fet4-1::LEU2),DY1455(MATa
Ade6 can1 his3 leu2 trp1 ura3) given by Agricultural University Of Nanjing Zhang Hongsheng professor's friendship.
Embodiment 1 corn zinc-iron regulates and controls the clone of transporter ZmZIPs gene
1st, the process of vegetable material
First vermiculite is impregnated with Hoagland nutrient solution, corn inbred line X178 seed point is sowed in seedlings nursing plate, above
Cover the dry vermiculite 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 nutrient 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
Quasi- nutrient solution and not zincification, iron, copper, manganese, Gao Xin, process 0 under conditions of iron, 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 corn total serum IgE
Corn 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 L25mM
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：
ZmZIP1F5'-GCGGCCGCATGCGCCGCCAAAGCCT-3'NotI
ZmZIP1R5'-GCGGCCGCTTATTCTACCAGAGAAATGCCTAGAGCG-3'NotI
ZmZIP2F5'-TACGTAATGGCCCGCGCCAC-3'SnaBI
ZmZIP2R 5'-TACGTATCAGGTGTCCCATATCATGACG-3'SnaBI
ZmZIP3F 5'-CCCGGGATGGGAGCTGTGAAGCATACATTG-3'SmaI
ZmZIP3R 5'-GGTACCCTATGCCCATATAGCAAGCATGGAC-3'KpnI
ZmZIP7F 5'-TCTAGAATGGTTCTCGCCGGCCTC-3'XbaI
ZmZIP7R 5'-GAGCTCTCAAGCCCATATTGCAAGTGATGACATAG-3'SacI
ZmZIP8F 5'-CCCGGGATGGCCATGAGGCCACG-3'SmaI
ZmZIP8R 5'-GAGCTCCTAGGCCCACTTGGCCAGC-3'SacI
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 denaturation 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 recombinant plasmid through digestion identification, sequencing is correctly cloned, and the 1st being cloned gene is ordered
Entitled ZmZIP1, the cDNA sequence of this gene is shown in SEQ ID No.1, and the amino acid sequence derived is SEQ ID No.2
Shown；The 2nd unnamed gene cloned is ZmZIP2, and the cDNA sequence of this gene is shown in SEQ ID No.3, is derived
Amino acid sequence is shown in SEQ ID No.4；The 3rd unnamed gene cloned is ZmZIP3, and the cDNA sequence of this gene is
Shown in SEQ ID No.5, the amino acid sequence derived is shown in SEQ ID No.6；The 4th unnamed gene cloned be
ZmZIP7, the cDNA sequence of this gene is shown in SEQ ID No.7, and the amino acid sequence derived is shown in SEQ ID No.8；
The 5th unnamed gene cloned is ZmZIP8, and the cDNA sequence of this gene is shown in SEQ ID No.9, the amino derived
Acid sequence is shown in SEQ ID No.10.
Expression pattern in seedling, embryo and endosperm for embodiment 2 ZmZIPs
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：
CDNA2.0 μ L, ExTaq0.1 μ L, 2 × GCI buffer solution 10.0 μ L, 10mM dNTP mix0.8 μ L, upstream primer
RTZmZIP1F/RTZmZIP2F/RTZmZIP3F/RTZmZIP7F/RTZmZIP8F (10 μM/μ L) 1.0 μ L, downstream primer
RTZmZIP1R/RTZmZIP2R/RTZmZIP3R/RTZmZIP7R/RTZmZIP8R (10 μM/μ L) 1.0 μ L, ddH2O5.1 μ L, always
Count 20.0 μ L；
RTZmZIP1F 5'-CCTCTCTGCGTTGGTTGCTCT-3'
RTZmZIP1R 5'-TTGATGGTTGTTTTCTGGTCGT-3'
RTZmZIP2F 5'-CCACAAATGGCACGAGGTCT-3'
RTZmZIP2R 5'-CGAAGACGGAGTGGAAGCAAA-3'
RTZmZIP3F 5'-GCCTCTTGTTGGTGCCCTTA-3'
RTZmZIP3R 5'-TCAACAATGAACGCTGTAGTGCT-3'
RTZmZIP7F 5'-ACTAGGTGGGTGCATTGCTCAG-3'
RTZmZIP7R 5'-TGCCAGCAGATACCGAGTCAA-3'
RTZmZIP8F 5'-CGTGTCATCGCTCAGGTTCTTG-3'
RTZmZIP8R 5'-CCCTCGAACATTTGGTGGAAG-3'
PCR reaction condition：94 DEG C of denaturations 4min；30 circulations, each 94 DEG C of denaturation 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 I10.0 μ L, Rox0.4
μ L, upstream primer ZmActin1F (10 μM/μ L) 0.4 μ L, downstream primer ZmActin1R (10 μM/μ L) 0.4 μ L, ddH2O3.8μ
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 computational chart
The amount of reaching.
Find, under the conditions of normal nutrition, 5 ZmZIPs genes are mainly on ground by real-time RT-PCR expression analysis
Top is expressed, and wherein, under the conditions of scarce zinc, ZmZIP8 is in 96h overground part up-regulated, ZmZIP3 overground part and underground part in 6h
Expression has all raised；Expression in high zink rod part ZmZIP7 and ZmZIP8 portion on the ground is gradually lowered, ZmZIP3
Significantly reduce in the expression of underground part.These results indicate that ZmZIP3, ZmZIP7 and ZmZIP8 in seedling period to zinc
Concentration is more sensitive.Under the conditions of iron deficiency, ZmZIP7 and ZmZIP8 is gradually increased with the expression of underground on the ground, explanation
ZmZIP7 and ZmZIP8 is more sensitive to the concentration of iron.Under the conditions of scarce copper, manganese deficiency, the expression of 5 ZmZIPs genes does not all have
Significantly change.5 ZmZIPs genes may participate in the growth (Fig. 2, Fig. 3 and Fig. 4) of embryo and endosperm.
The bioinformatic analysis of embodiment 3 ZmZIPs
ZmZIPs is made up of 367-483 amino acid, containing 6-9 membrane spaning domain, between the 3rd and the 4th transmembrane region
There is a variable region being rich in histidine, may be relevant with the binding transport of metal ion.Phylogenetic analysis show, ZmZIP1 with
AtIAR1, OsIAR1 evolutionary relationship is nearer.In addition, ZmZIP3 and ZmZIP4 and OsZIP3 and OsZIP4 forms a gene cluster,
ZmZIP2 is neighbouring with OsZIP2, and zinc transporter OsZIP1, AtZIP2 and AtZIP11 in a branch, ZmZIP5 with
, in a branch, ZmZIP8 and OsZIP8, ZmZIP6 and OsZIP6 evolutionary relationship is nearer for ZmZIP7, and these results show, this
Inventing 5 separated ZmZIPs is probably zinc-iron transporter.
The Subcellular Localization of embodiment 4 ZmZIPs
1st, the structure of fusion expression vector
According to the primers of ZmZIPs gene, primer sequence is as follows：
ZmZIP1GF 5'-GAATTCATGCGCCGCCAAAGCCT-3'EcoRI
ZmZIP1GR 5'-TCTAGATTCTACCAGAGAAATGCCTAGAGCG-3'XbaI
ZmZIP2GF 5'-GAATTCATGGCCCGCGCCACCAA-3'EcoRI
ZmZIP2GR 5'-TCTAGAGGTGTCCCATATCATGACGACGG-3'XbaI
ZmZIP3GF 5'-GAATTCATGGGAGCTGTGAAGCATAC-3'EcoRI
ZmZIP3GR 5'-TCTAGATGCCCATATAGCAAGCATGGACAT-3'XbaI
ZmZIP7GF 5'-GAATTCATGGTTCTCGCCGGCCTC-3'EcoRI
ZmZIP7GR 5'-TCTAGAAGCCCATATTGCAAGTGATGACATAG-3'XbaI
ZmZIP8GF 5'-GAATTCATGGCCATGAGGCCACGC-3'EcoRI
ZmZIP8GR 5'-TCTAGAGGCCCACTTGGCCAGCAT-3'XbaI
Add suitable restriction enzyme site, and gene 3 ' end removes terminator 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 denaturation 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 electrophoresis, convert coli strain Mach1-T1,
Obtain positive colony, upgrading grain, digestion 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 electrophoresis, after the correct plasmid enzyme restriction that is sequenced, by purpose piece
Section be building up on pRTL2NGFP carrier, be respectively designated as pRTL2NGFP-ZmZIP1, pRTL2NGFP-ZmZIP2,
PRTL2NGFP-ZmZIP3, pRTL2NGFP-ZmZIP7, pRTL2NGFP-ZmZIP8, Fig. 5 is digestion qualification figure.
2nd, with corresponding digestion pRTL2NGFP carrier and the genetic fragment after different digestions, through T4DNA ligase is even
Connect, convert 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 5 ZmZIPs (ZmZIP1, ZmZIP2, ZmZIP3, ZmZIP7 or ZmZIP8) are both positioned at from positioning result
Plasma membrane and (Fig. 6) on intercellular membrane.For further determining that the concrete positioning of intercellular membrane, from ER marker and ZmZIPs altogether
Positioning carries out the conversion of arabidopsis mesophyll protoplast, and result proves that 5 ZmZIPs are both positioned in cytoplasma membrane and endoplasmic reticulum
(Fig. 7).
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：
ZmZIP1YF 5'-GCGGCCGCATGCGCCGCCAAAGCCT-3'NotI
ZmZIP1YR 5'-GCGGCCGCTTATTCTACCAGAGAAATGCCTAGAGCG-3'NotI
ZmZIP2YF 5'-TACGTAATGGCCCGCGCCAC-3'SnaBI
ZmZIP2YR 5'-TACGTATCAGGTGTCCCATATCATGACG-3'SnaBI
ZmZIP3YF 5'-CCCGGGATGGGAGCTGTGAAGCATACATTG-3'SmaI
ZmZIP3YR 5'-GGTACCCTATGCCCATATAGCAAGCATGGAC-3'KpnI
ZmZIP7YF 5'-TACGTAATGGTTCTCGCCGGCCTC-3'SnaBI
ZmZIP7YR 5'-TACGTATCAAGCCCATATTGCAAGTGATGACATAG-3'SnaBI
ZmZIP8YF 5'-TGCCATGGCCATGAGGCCAC-3'
ZmZIP8YR 5'-CTAGGCCCACTTGGCCAGCATG-3'
OsZIP5YF 5'-CCCGGGGAGCCATCGGCGATGGCGA-3'SmaI
OsZIP5YR 5'-GAGCTCGTGATGGTCACTCACTCATCACGCC-3'SacI
OsZIP8YF 5'-GCGGCCGCATGAGGACGAACACCACC-3'NotI
OsZIP8YR 5'-GCGGCCGCCCTCTACATTAGTCCCTGAG-3'NotI
OsIRT1YF 5'-GCGGCCGCCCCGGGATGGCGACGCCGCGGA-3'NotI,SmaI
OsIRT1YR 5'-GCGGCCGCCCCGGGTCACGCCCACTTGGCCATG-3'NotI,SmaI
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 denaturation 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 electrophoresis
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 after correct plasmid enzyme restriction, purpose fragment be building up on pFL61 carrier, be named as pFL61-
ZmZIP1, pFL61-ZmZIP2, pFL61-ZmZIP3, pFL61-ZmZIP7, pFL61-ZmZIP8 and pFL61-OsZIP5,
PFL61-OsZIP8 and pFL61-OsIRT1, Fig. 8 are digestion qualification figure.
With the ZmZIPs fragment after NotI digestion pFL61 carrier and digestion 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 nutrient medium in, 28 DEG C of shaking table culture about 24h；
(2) bacterium solution, drawing above 2% volume is transferred to the numerous about 4-5h of continuation expansion 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, packing；
(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 precooling, must not have bubble；
(10), electroporated, add about 800 μ L, the sorbitol solution of the precooling 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 vitellophag 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 absolute ethyl 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 wash-outs in the middle part of adsorbed film
Buffer solution 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% glycerine to preserve in -80 DEG C.
4th, yeast complementation experiment result
By pFL61, pFL61-ZmZIP1, pFL61-ZmZIP2, pFL61-ZmZIP3, pFL61-ZmZIP7, pFL61-
ZmZIP8, pFL61-OsZIP5, pFL61-OsZIP8, pFL61-OsIRT1 plasmid is transformed into yeast mutant respectively
In 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 biology2010,73(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.) for 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.) for the positive control of iron transporters, pFL61 converts wild-type strain DY1455
As another positive control, the saccharomycete being accredited as the positive after conversion is cultivated in SD fluid nutrient medium, and yeast liquid is dilute respectively
Release 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 iron and normal SD, low
(SD culture medium adds 0.4mM EDTA, 0.4mM EDTA and 250 μM of ZnSO to zinc culture medium4, 0.4mM EDTA and 300 μM
ZnSO4), (SD culture medium adds 50mM MES, 50mM MES and 50 μM of FeCl to low iron 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 phytologist2009,182(2):392-404.) method is carried out, 28 DEG C of cultures, observes within 6 days
Result of the test.
Experimental result shows, under the conditions of low zinc, added with 250 μM of ZnSO4Culture medium in can substantially observe DY-
PFL61 (wild type), Z-ZmZIP1, Z-ZmZIP2, Z-ZmZIP3, Z-ZmZIP7, Z-ZmZIP8, Z-OsZIP5, Z-OsZIP8,
Z-OsIRT1 is better than empty carrier Z-pFL61 growing way, and, Z-ZmZIP1, Z-ZmZIP2, Z-ZmZIP3, Z-ZmZIP7, Z-
ZmZIP8 with it has been reported that rice Os ZIP growing way suitable.Under the conditions of low iron, D-ZmZIP1, D-ZmZIP2, D-ZmZIP3,
D-ZmZIP7, D-ZmZIP8 are better than empty carrier D-pFL61 growing way, but do not have it has been reported that D-OsIRT1 transport activity strong
(Fig. 9)；No matter ZmZIP1, ZmZIP2, ZmZIP3, ZmZIP7 or ZmZIP8 show not in low zinc or under the conditions of low iron
With the transport activity of degree, illustrate that ZmZIP1, ZmZIP2, ZmZIP3, ZmZIP7 or ZmZIP8 have the function of transhipment zinc-iron.
Experimental example 1 ZmZIPs gene overexpression in arabidopsis improves the experiment of iron and Zn content in arabidopsis seed
ZmZIP1, ZmZIP2, ZmZIP3, ZmZIP7 and ZmZIP8 gene is controlled with composing type 35S promoter respectively
The plant expression vector pBI121 structure that is connected obtain ZmZIP1, ZmZIP2, ZmZIP3, ZmZIP7 and ZmZIP8 genetic recombination
Plant expression vector (Figure 10)；The recombinant plant expression vector of structure is transformed in arabidopsis respectively, identification obtains positive
Turn ZmZIP1, ZmZIP2, ZmZIP3, ZmZIP7 and ZmZIP8 gene arabidopsis；By positive turn ZmZIPs gene arabidopsis with
Wild type Colombia cultivates under the conditions of identical culture, harvests and turns ZmZIPs gene arabidopsis seed and wild type seeds,
Measure the content turning iron or zinc in ZmZIPs gene arabidopsis seed and wildtype Arabidopsis thaliana seed respectively；Weigh a certain amount of kind
Sub- material, through micro-wave digestion, constant volume, carries out the mensure of zinc iron content with ICP-MS method；Every batch of mensure 200mg seed, measures three
Batch, take the mean value of three batch datas.Measurement result is shown in Figure 11.From the result of Figure 11, in arabidopsis overexpression ZmZIP1,
ZmZIP2, ZmZIP3, ZmZIP7 or ZmZIP8 gene all can be different degrees of raising seed in iron or Zn content.
Claims (7)
1. from corn (Zea mays) detached zinc-iron regulation and control transporter ZmZIP1 gene it is characterised in that its cDNA sequence
Shown in SEQ ID No.1.
2. the protein of ZmZIP1 gene code described in claim 1 is it is characterised in that the amino acid sequence of described protein is
Shown in SEQ ID No.2.
3. the recombinant expression carrier of transporter ZmZIP1 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 ZmZIP1 gene is improving plant absorption, transhipment or storage zinc or iron energy
Application in power.
6. zinc-iron described in claim 1 regulates and controls transporter ZmZIP1 gene in the zinc releasing excess or iron in plant murder by poisoning
Application.
7. according to the application described in claim 5 or 6 it is characterised in that including：By zinc-iron regulation and control transhipment described in claim 1
Body ZmZIP1 gene is exercisable to be connected with expression regulation element, obtains recombinant plant expression vector；Recombinant plant is expressed
Carrier transformation receptor plant or plant cell, cultivate and obtain genetically modified plants.
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| CN201410240908.XA CN104212816B (en) | 2013-05-31 | 2014-05-30 | Maize zinc-iron regulation transporter ZmZIPs gene and application thereof |
| CN201611228703.5A CN106434698A (en) | 2013-05-31 | 2014-05-30 | Maize zinc-iron regulation transporter ZmZIP3 gene and application thereof |
| CN201611228704.XA CN106754962A (en) | 2013-05-31 | 2014-05-30 | Maize zinc-iron regulation transporter ZmZIP8 gene and application thereof |
| CN201611229682.9A CN106636134A (en) | 2013-05-31 | 2014-05-30 | Maize zinc-iron regulation transporter ZmZIP2 gene and application thereof |
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| CN201611229682.9A Division CN106636134A (en) | 2013-05-31 | 2014-05-30 | Maize zinc-iron regulation transporter ZmZIP2 gene and application thereof |
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| CN106754969A (en) * | 2017-01-23 | 2017-05-31 | 内蒙古和盛生态育林有限公司 | A kind of gene for promoting plant absorption zinc ion, amino acid sequence and expression vector and application |
| CN110117596A (en) * | 2019-04-30 | 2019-08-13 | 山东农业大学 | A kind of corn seed grouting controlling gene ZmDef1 and application |
| CN116121434B (en) * | 2022-09-26 | 2023-12-12 | 四川农业大学 | Amino acid locus and molecular marker for controlling wheat ZIP3A zinc transport function |
| CN115976056B (en) * | 2023-01-10 | 2024-03-12 | 南京农业大学 | Application of rice zinc-iron regulatory transporter encoding gene OsZIP2 |
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| US6590140B1 (en) * | 1996-05-29 | 2003-07-08 | Trustees Of Dartmouth College | Metal-regulated transporters and uses therefor |
| CN101892243A (en) * | 2010-01-29 | 2010-11-24 | 首都师范大学 | Complementary deoxyribonucleic acid (cDNA) sequence related to iron deficiency of plant and encoded protein and application thereof |
| CN102498125A (en) * | 2009-07-16 | 2012-06-13 | 瓦赫宁恩大学 | Regulation of zinc deficiency and tolerance in plants |
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| CN102304526B (en) * | 2011-09-28 | 2013-08-21 | 首都师范大学 | Application of Malus xiaojinensis Cheng et Jiang Fe-regulation transporter gene in improvement of Fe content in plant |
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| CN102498125A (en) * | 2009-07-16 | 2012-06-13 | 瓦赫宁恩大学 | Regulation of zinc deficiency and tolerance in plants |
| CN101892243A (en) * | 2010-01-29 | 2010-11-24 | 首都师范大学 | Complementary deoxyribonucleic acid (cDNA) sequence related to iron deficiency of plant and encoded protein and application thereof |
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