CN1289523C - Paddy rice potassium, sodium ion transport gene and its application - Google Patents

Abstract

The present invention provides a new plant gene-paddy rice K<+>/Na<+> transport protein and a coding protein thereof. The present invention also discloses an application of the K<+>/Na<+> transport protein gene, and especially the application for changing the salt resistance of plants in the breed improvement and crossbreeding of plants.

Description

Paddy rice potassium, sodium ion transporter gene and application thereof

Technical field

The invention belongs to the phytology field, specifically, the present invention relates to the K of new paddy rice (Oryza sativa) ⁺/ Na ⁺Transporter gene and proteins encoded thereof.The invention also discloses this K of coding ⁺/ Na ⁺The purposes of the gene of translocator especially is used to change the salt tolerance of plant in plant species improvement and cross-breeding.

Background technology

Potassium (K) is one of plant most important three big a large amount of essential elements.Potassium ion (K ⁺) be one of positively charged ion that content is the highest in the vegetable cell, it can regulate many important physiological function of plant materials, as improving plant salt endurance, strengthen photosynthesis of plants, strengthening the synthetic and transhipment of plant substance in vivo etc.Therefore study controlling plant K ⁺It is very important that the molecular mechanism of ionic absorption seems, also is the focus of Recent study.

Plant K ⁺Mainly by channel protein gene and transporter gene control, the former is essentially passive transportation to ionic absorption, and the latter is an active transport.Channel protein gene mainly is two big class AKT and KAT, and transporter gene mainly contains HKT (High-Affinity K ⁺Transporter, high-affinity kalium ion transport albumen) and HAK (High-Affinity K ⁺Uptake, high-affinity potassium ion picked-up albumen) two big classes.

Studies show that in recent years, some transporter gene also has transportation Na ⁺Function, thereby closely related with plant salt endurance.At present, in paddy rice clone and functional analysis OsHKT1 and OsHKT4 gene.The result shows the only high affine transportation Na of OsHKT1 ⁺, and OsHKT4 is to K ⁺/ Na ⁺Transportation simultaneously.Because the result of the long-term evolution of plant has formed complicated ionic absorption mechanism, the gene that relates to is more.

Because K ⁺/ Na ⁺The salt tolerance of transporter gene and plant is closely related, and therefore, in order to change the salt tolerance of plant variety effectively, pointedly, this area presses for exploitation and salt tolerance proteins associated and encoding gene thereof.

Summary of the invention

An object of the present invention is to provide a kind of new K ⁺/ Na ⁺Translocator (is paddy rice K ⁺/ Na ⁺Translocator) and fragment, analogue and derivative.

Another object of the present invention provides the polynucleotide of these polypeptide of coding.

Another object of the present invention provides method and the purposes of this polypeptide and encoding sequence, the especially purposes aspect the change plant salt endurance of producing these polypeptide.

In a first aspect of the present invention, provide novel isolated paddy rice K ⁺/ Na ⁺Translocator, this peptide source is from paddy rice, and it comprises: polypeptide or its conservative property variation polypeptide or its active fragments or its reactive derivative with SEQ ID NO:2 aminoacid sequence.Preferably, this polypeptide is selected from down group: the polypeptide that (a) has SEQ ID NO:2 aminoacid sequence; (b) replacement, disappearance or the interpolation of SEQ ID NO:2 aminoacid sequence through one or more amino-acid residues formed, and have the plant of promotion K ⁺/ Na ⁺Transport function by (a) polypeptides derived.

In a second aspect of the present invention, the polynucleotide of isolating these polypeptide of coding are provided, these polynucleotide comprise a nucleotide sequence, and this nucleotide sequence is shown at least 70% homogeny with a kind of nucleotides sequence that is selected from down group: (a) the above-mentioned paddy rice K of coding ⁺/ Na ⁺The polynucleotide of translocator; (b) with polynucleotide (a) complementary polynucleotide.Preferably, this polynucleotide encoding has the polypeptide of aminoacid sequence shown in the SEQ ID NO:2.More preferably, the sequence of these polynucleotide is be selected from down group a kind of:

(a) has the sequence of 1-1665 position among the SEQ ID NO:1;

(b) has the sequence of 1-1662 position among the SEQ ID NO:1;

(c) has the nucleotide sequence of 1-3999 position among the SEQ ID NO:3.。

In a third aspect of the present invention, the carrier that contains above-mentioned polynucleotide is provided, and has been transformed or host cell of transduceing or the host cell that is directly transformed or transduce by above-mentioned polynucleotide by this carrier.

In a fourth aspect of the present invention, provide preparation paddy rice K ⁺/ Na ⁺The method of translocator, this method comprises: (a) under conditions suitable for the expression, cultivate the above-mentioned host cell that is transformed or transduce; (b) from culture, isolate paddy rice K ⁺/ Na ⁺Translocator.

In a fifth aspect of the present invention, provide and above-mentioned paddy rice K ⁺/ Na ⁺Translocator specificity bonded antibody.The nucleic acid molecule that can be used for detecting also is provided, and it contains a successive 15-1500 Nucleotide in the above-mentioned polynucleotide.

In a sixth aspect of the present invention, provide in the proteic antibody of anti-the present invention and a kind of test sample whether have paddy rice K ⁺/ Na ⁺The method of translocator, it comprises: with sample and paddy rice K ⁺/ Na ⁺The specific antibody contact of translocator observes whether form antibody complex, has formed antibody complex and has just represented to exist in the sample paddy rice K ⁺/ Na ⁺Translocator.

In a seventh aspect of the present invention, a kind of method that changes plant salt endurance is provided, it comprises step:

(1) provide the Agrobacterium of carrying expression vector, described expression vector contains paddy rice ^K/ Na ⁺Translocator dna encoding sequence, described paddy rice K ⁺/ Na ⁺Translocator is selected from down group:

(a) has the polypeptide of SEQ ID NO:2 aminoacid sequence;

(b) replacement, disappearance or the interpolation of SEQ ID NO:2 aminoacid sequence through one or more amino-acid residues formed, and have the plant of promotion K ⁺/ Na ⁺Transport function by (a) polypeptides derived;

(2) vegetable cell or tissue or organ are contacted with Agrobacterium in the step (1), thereby make paddy rice K ⁺/ Na ⁺Translocator dna encoding sequence changes vegetable cell over to, and is incorporated on the karyomit(e) of vegetable cell;

(3) select and change paddy rice K over to ⁺/ Na ⁺The vegetable cell of translocator dna encoding sequence or tissue or organ;

(4) vegetable cell in the step (3) or tissue or neomorph are become plant.

Others of the present invention are because disclosing of the technology of this paper is conspicuous to those skilled in the art.

Description of drawings

Fig. 1 has shown K under the control paddy rice salt stress ⁺/ Na ⁺The aminoacid sequence of transporter gene SKc1 and with HKT gene family member's homology relatively.

Fig. 2 has shown the near isogenic line (SKc-NIL) of (140mM NaCl) paddy rice SKc1 and the Na of recurrent parent " light more " under the salt stress ⁺Concentration.

Embodiment

The inventor from paddy rice (Oryza sativa.L), utilizes the map based cloning method to be cloned into control paddy rice K under salt stress in conjunction with bioinformatics method from height salt water resistance rice varieties through extensive and deep research first ⁺/ Na ⁺The new gene of ionic absorption, and it is carried out functional study, show that this gene obviously increases K under salt stress ⁺Absorb and reduction Na ⁺, reduce ion and poison, and then can strengthen the salt tolerance of paddy rice, therefore in the breeding of farm crop resistance, have application prospect.

In the present invention, term " paddy rice K ⁺/ Na ⁺Translocator ", " paddy rice K ⁺/ Na ⁺The transhipment polypeptide ", " SKc1 albumen ", " SKc1 polypeptide " etc. are used interchangeably, and all refer to have paddy rice K ⁺/ Na ⁺The albumen or the polypeptide of translocator aminoacid sequence (SEQ ID NO:2).

When not particularly pointing out, term " K ⁺/ Na ⁺Translocator " comprise wild-type and mutant K ⁺/ Na ⁺Translocator.

As used herein, " isolating " is meant that material separates (if natural substance, primal environment promptly is a natural surroundings) from its primal environment.Do not have separation and purification as polynucleotide under the native state in the active somatic cell and polypeptide, but same polynucleotide or polypeptide as from native state with in other materials that exist separately, then for separation and purification.

As used herein, " isolating paddy rice K ⁺/ Na ⁺Translocator or polypeptide " be meant paddy rice K ⁺/ Na ⁺Translocator is substantially free of natural relative other albumen, lipid, carbohydrate or other material.Those skilled in the art can use the purified technology of protein purifying paddy rice K of standard ⁺/ Na ⁺Translocator.Basically pure polypeptide can produce single master tape on non-reduced polyacrylamide gel.

Polypeptide of the present invention can be recombinant polypeptide, natural polypeptides, synthetic polypeptide, preferred recombinant polypeptide.Polypeptide of the present invention can be the product of natural purifying, or the product of chemosynthesis, or uses recombinant technology to produce from protokaryon or eucaryon host (for example, bacterium, yeast, higher plant, insect and mammalian cell).The host used according to the recombinant production scheme, polypeptide of the present invention can be glycosylated, maybe can be nonglycosylated.Polypeptide of the present invention also can comprise or not comprise initial methionine residues.

The present invention also comprises paddy rice K ⁺/ Na ⁺The fragment of translocator, derivative and analogue.As used herein, term " fragment ", " derivative " and " analogue " are meant and keep natural paddy rice K of the present invention basically ⁺/ Na ⁺Biological function that translocator is identical or active polypeptide.Polypeptide fragment of the present invention, derivative or analogue can be that (i) has one or more conservative or substituted polypeptide of non-conservation amino-acid residue (preferred conservative amino acid residue), and the amino-acid residue of such replacement can be also can not encoded by genetic code, or (ii) in one or more amino-acid residues, has a polypeptide of substituted radical, or (iii) mature polypeptide and another compound (such as the compound that prolongs the polypeptide transformation period, polyoxyethylene glycol for example) merges formed polypeptide, or (iv) additional aminoacid sequence is fused to this peptide sequence and the polypeptide that forms (as leader sequence or secretion sequence or be used for the sequence or the proteinogen sequence of this polypeptide of purifying or fusion rotein).According to the instruction of this paper, these fragments, derivative and analogue belong to the known scope of those skilled in the art.

In the present invention, term " paddy rice K ⁺/ Na ⁺Translocator " refer to have paddy rice K ⁺/ Na ⁺The active SEQ ID of translocator NO.3 polypeptide of sequence.This term also comprises having and paddy rice K ⁺/ Na ⁺The variant form translocator identical function, SEQ ID NO.3 sequence.These variant forms comprise (but being not limited to): one or more (it is individual to be generally 1-50, preferably 1-30, more preferably 1-20,1-10 best) amino acid whose disappearance, insertion and/or replacement, and add one or several at C-terminal and/or N-terminal and (be generally in 20, preferably being in 10, more preferably is in 5) amino acid.For example, in the art, when replacing, can not change proteinic function usually with the close or similar amino acid of performance.Again such as, add one or several amino acid at C-terminal and/or N-terminal and also can not change proteinic function usually.This term also comprises paddy rice K ⁺/ Na ⁺The active fragments of translocator and reactive derivative.

The variant form of this polypeptide comprises: homologous sequence, conservative property varient, allelic variant, natural mutation, induced mutation body, under high or low tight degree condition can with paddy rice K ⁺/ Na ⁺The albumen that the DNA of translocator DNA hybridization is coded and utilize anti-paddy rice K ⁺/ Na ⁺Polypeptide or albumen that the antiserum(antisera) of translocator obtains.The present invention also provides other polypeptide, as comprises paddy rice K ⁺/ Na ⁺Translocator or its segmental fusion rotein.Except the polypeptide of total length almost, the present invention has also comprised paddy rice K ⁺/ Na ⁺The soluble fragments of translocator.Usually, this fragment has paddy rice K ⁺/ Na ⁺The translocator sequence at least about 10 continuous amino acids, usually at least about 30 continuous amino acids, preferably at least about 50 continuous amino acids, more preferably at least about 80 continuous amino acids, best at least about 100 continuous amino acids.

Invention also provides paddy rice K ⁺/ Na ⁺The analogue of translocator or polypeptide.These analogues and natural paddy rice K ⁺/ Na ⁺The difference of translocator can be the difference on the aminoacid sequence, also can be the difference that does not influence on the modified forms of sequence, perhaps haves both at the same time.These polypeptide comprise natural or the inductive genetic variant.The induce variation body can obtain by various technology, as by radiation or be exposed to mutagenic compound and produce random mutagenesis, also can pass through site-directed mutagenesis method or the biological technology of other known moleculars.Analogue also comprises having the analogue that is different from the amino acid whose residue of natural L-(as D-amino acid), and has non-natural analogue that exist or synthetic amino acid (as β, gamma-amino acid).Should be understood that polypeptide of the present invention is not limited to the above-mentioned representational polypeptide that exemplifies.

(the not changing primary structure usually) form of modification comprises: the chemically derived form such as the acetylize or carboxylated of the polypeptide that body is interior or external.Modify and also comprise glycosylation.Modified forms also comprises have the phosphorylated amino acid residue sequence of (as Tyrosine O-phosphate, phosphoserine, phosphothreonine).Thereby also comprise the polypeptide that has been improved its anti-proteolysis performance or optimized solubility property by modifying.

In the present invention, " paddy rice K ⁺/ Na ⁺Translocator conservative property variation polypeptide " refer to compare with the aminoacid sequence of SEQ ID NO:2, there are 10 at the most, preferably at the most 8, more preferably at the most 5,3 amino acid is replaced by similar performance or close amino acid and is formed polypeptide at the most best.These conservative property variation polypeptide preferably carry out the amino acid replacement according to table 1 and produce.

Table 1

Initial residue	Representational replacement	The preferred replacement
			Ala(A)	Val；Leu；Ile	Val
Arg(R)	Lys；Gln；Asn	Lys
			Asn(N)	Gln；His；Lys；Arg	Gln
Asp(D)	Glu	Glu
			Cys(C)	Ser	Ser
Gln(Q)	Asn	Asn
			Glu(E)	Asp	Asp
Gly(G)	Pro；Ala	Ala
			His(H)	Asn；Gln；Lys；Arg	Arg
Ile(I)	Leu；Val；Met；Ala；Phe	Leu
			Leu(L)	Ile；Val；Met；Ala；Phe	Ile
Lys(K)	Arg；Gln；Asn	Arg
			Met(M)	Leu；Phe；Ile	Leu
Phe(F)	Leu；Val；Ile；Ala；Tyr	Leu
			Pro(P)	Ala	Ala
Ser(S)	Thr	Thr
			Thr(T)	Ser	Ser
Trp(W)	Tyr；Phe	Tyr
			Tyr(Y)	Trp；Phe；Thr；Ser	Phe
Val(V)	Ile；Leu；Met；Phe；Ala	Leu

Polynucleotide of the present invention can be dna form or rna form.Dna form comprises the DNA of cDNA, genomic dna or synthetic.DNA can be strand or double-stranded.DNA can be coding strand or noncoding strand.The coding region sequence of encoding mature polypeptide can be identical with the coding region sequence shown in the SEQ ID NO:1 or the varient of degeneracy.As used herein, " varient of degeneracy " is meant that in the present invention coding has the protein of SEQ ID NO:2, but with the differentiated nucleotide sequence of coding region sequence shown in the SEQ ID NO:1.

The polynucleotide of the mature polypeptide of coding SEQ ID NO:2 comprise: the encoding sequence of an encoding mature polypeptide; The encoding sequence of mature polypeptide and various additional code sequence; Encoding sequence of mature polypeptide (with optional additional code sequence) and non-coding sequence.

Term " polynucleotide of coded polypeptide " can be the polynucleotide that comprise this polypeptide of encoding, and also can be the polynucleotide that also comprise additional code and/or non-coding sequence.

The invention still further relates to the varient of above-mentioned polynucleotide, its coding has the polypeptide of identical aminoacid sequence or fragment, analogue and the derivative of polypeptide with the present invention.The varient of these polynucleotide can be the allelic variant of natural generation or the varient that non-natural takes place.These nucleotide diversity bodies comprise and replace varient, deletion mutation body and insert varient.As known in the art, allelic variant is the replacement form of polynucleotide, and it may be replacement, disappearance or the insertion of one or more Nucleotide, but can be from not changing the function of its encoded polypeptides in fact.

The invention still further relates to and above-mentioned sequence hybridization and two sequences between have at least 50%, preferably at least 70%, the polynucleotide of at least 80% homogeny more preferably.The present invention be more particularly directed under stringent condition and the interfertile polynucleotide of polynucleotide of the present invention.In the present invention, " stringent condition " is meant: (1) than hybridization under low ionic strength and the comparatively high temps and wash-out, as 0.2 * SSC, and 0.1%SDS, 60 ℃; Or (2) hybridization the time is added with denaturing agent, as 50% (v/v) methane amide, 0.1% calf serum/0.1%Ficoll, 42 ℃ etc.; Or (3) only at the homogeny between the two sequences at least more than 90%, be more preferably 95% and just hybridize when above.And the polypeptide of interfertile polynucleotide encoding has identical biological function and activity with the mature polypeptide shown in the SEQ ID NO:2.

The invention still further relates to nucleic acid fragment with above-mentioned sequence hybridization.As used herein, the length of " nucleic acid fragment " contains 15 Nucleotide at least, better is at least 30 Nucleotide, is more preferably at least 50 Nucleotide, preferably more than at least 100 Nucleotide.Nucleic acid fragment can be used for the amplification technique (as PCR) of nucleic acid to determine and/or separation coding paddy rice K ⁺/ Na ⁺The polynucleotide of translocator.

Paddy rice K of the present invention ⁺/ Na ⁺Translocator Nucleotide full length sequence or its fragment can obtain with the method for pcr amplification method, recombination method or synthetic usually.For the pcr amplification method, can be disclosed according to the present invention about nucleotide sequence, especially open reading frame sequence designs primer, and with commercially available cDNA storehouse or by the prepared cDNA storehouse of ordinary method well known by persons skilled in the art as template, amplification and must relevant sequence.When sequence is longer, usually needs to carry out twice or pcr amplification repeatedly, and then the fragment that each time amplifies is stitched together by proper order.

In case obtained relevant sequence, just can obtain relevant sequence in large quantity with recombination method.This normally is cloned into carrier with it, changes cell again over to, separates obtaining relevant sequence then from the host cell after the propagation by ordinary method.

In addition, also the method for available synthetic is synthesized relevant sequence, especially fragment length more in short-term.Usually, by first synthetic a plurality of small segments, and then connect and to obtain the very long fragment of sequence.

At present, can be fully obtain the dna sequence dna of code book invention albumen (or its fragment, or derivatives thereof) by chemosynthesis.This dna sequence dna can be introduced in various existing dna moleculars as known in the art (or as carrier) and the cell then.In addition, also can will suddenly change and introduce in the protein sequence of the present invention by chemosynthesis.

The present invention also relates to comprise the carrier of polynucleotide of the present invention, and with carrier of the present invention or paddy rice K ⁺/ Na ⁺The host cell that transport protein coding sequence produces through genetically engineered, and the method that produces polypeptide of the present invention through recombinant technology.

Recombinant DNA technology (Science, 1984 by routine; 224:1431), can utilize polymerized nucleoside acid sequence of the present invention to can be used to express or produce the paddy rice K of reorganization ⁺/ Na ⁺Translocator.In general following steps are arranged:

(1). with coding paddy rice K of the present invention ⁺/ Na ⁺The polynucleotide of translocator (or varient), or transform or the transduction proper host cell with the recombinant expression vector that contains these polynucleotide;

(2). the host cell of in suitable medium, cultivating;

(3). separation, protein purification from substratum or cell.

Among the present invention, paddy rice K ⁺/ Na ⁺The translocator polynucleotide sequence can be inserted in the recombinant expression vector.Term " recombinant expression vector " refers to bacterial plasmid well known in the art, phage, yeast plasmid, vegetable cell virus, mammalian cell virus or other carriers.In a word, as long as can duplicate in host and stablize, any plasmid and carrier can be used.A key character of expression vector is to contain replication orgin, promotor, marker gene and translation controlling elements usually.

Method well-known to those having ordinary skill in the art can be used for structure and contain paddy rice K ⁺/ Na ⁺Translocator DNA sequences encoding and suitable transcribing/the translate expression vector of control signal.These methods comprise extracorporeal recombinant DNA technology, DNA synthetic technology, the interior recombinant technology of body etc.Described dna sequence dna can effectively be connected on the suitable promotor in the expression vector, and is synthetic to instruct mRNA.Expression vector also comprises ribosome bind site and the transcription terminator that translation initiation is used.

In addition, expression vector preferably comprises one or more selected markers, to be provided for selecting the phenotypic character of transformed host cells, cultivate Tetrahydrofolate dehydrogenase, neomycin resistance and the green fluorescent protein (GFP) of usefulness as eukaryotic cell, or be used for colibacillary tsiklomitsin or amicillin resistance.

Comprise the carrier of above-mentioned suitable dna sequence dna and suitable promotor or control sequence, can be used to transform appropriate host cell, so that it can marking protein.

Host cell can be a prokaryotic cell prokaryocyte, as bacterial cell; Or eukaryotic cell such as low, as yeast cell; Or higher eucaryotic cells, as vegetable cell.Representative example has: intestinal bacteria, streptomyces, Agrobacterium; Fungal cell such as yeast; Vegetable cell etc.

When polynucleotide of the present invention are expressed in higher eucaryotic cells, be enhanced if will make to transcribe when in carrier, inserting enhancer sequence.Enhanser is the cis acting factor of DNA, and nearly 10 to 300 base pairs act on promotor transcribing with enhancing gene usually.

Persons skilled in the art all know how to select appropriate carriers, promotor, enhanser and host cell.

Can carry out with routine techniques well known to those skilled in the art with the recombinant DNA transformed host cell.When the host was prokaryotic organism such as intestinal bacteria, the competent cell that can absorb DNA can be used CaCl in exponential growth after date results ₂Method is handled, and used step is well-known in this area.Another kind method is to use MgCl ₂If desired, transforming also the method for available electroporation carries out.When the host is an eukaryote, can select following DNA transfection method for use: coprecipitation of calcium phosphate method, conventional mechanical method such as microinjection, electroporation, liposome packing etc.

Transform plant and also can use methods such as Agrobacterium-mediated Transformation or particle gun conversion, for example leaf dish method.Can use ordinary method regeneration plant for plant transformed cell, tissue or organ, thereby obtain the plant that salt tolerance changes.

The transformant that obtains can be cultivated with ordinary method, expresses the polypeptide of coded by said gene of the present invention.According to used host cell, used substratum can be selected from various conventional substratum in the cultivation.Under the condition that is suitable for the host cell growth, cultivate.After host cell grows into suitable cell density, induce the promotor of selection with suitable method (as temperature transition or chemical induction), cell is cultivated for some time again.

The extracellular can be expressed or be secreted into to recombinant polypeptide in the above methods in cell or on cytolemma.If desired, can utilize its physics, the separating by various separation methods with other characteristic and the albumen of purification of Recombinant of chemistry.These methods are well-known to those skilled in the art.The example of these methods includes, but are not limited to: conventional renaturation handles, handle the combination of (salt analysis method), centrifugal, the broken bacterium of infiltration, uf processing, super centrifugal, sieve chromatography (gel-filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography (LC) technology and these methods with protein precipitant.

The paddy rice K of reorganization ⁺/ Na ⁺Translocator or polypeptide are of use in many ways.For example be used for screening promotion or antagonism paddy rice K ⁺/ Na ⁺The antibody of translocator function, polypeptide or other part.With the reorganization paddy rice K that expresses ⁺/ Na ⁺Translocator screening peptide library can be used for seeking valuable can the inhibition or stimulation paddy rice K ⁺/ Na ⁺The peptide molecule of translocator function.

On the other hand, the present invention also comprises paddy rice K ⁺/ Na ⁺Translocator DNA or the polypeptide of its fragment coding has specific polyclonal antibody and monoclonal antibody, especially monoclonal antibody.The present invention not only comprises complete mono-clonal or polyclonal antibody, but also comprises having immunocompetent antibody fragment or chimeric antibody.

Antibody of the present invention can be prepared by the known various technology of those skilled in that art.For example, the paddy rice K of purifying ⁺/ Na ⁺Transporter gene product or its have antigenic fragment, can be applied to animal to induce the generation of polyclonal antibody.Similarly, express paddy rice K ⁺/ Na ⁺Translocator or its have antigenic segmental cell and can be used to immune animal and produce antibody.This type of monoclonal antibody can utilize hybridoma technology to prepare.Each antibody-like of the present invention can utilize paddy rice K ⁺/ Na ⁺The fragment of transporter gene product or functional zone obtain by the routine immunization technology.These fragments or functional zone can utilize recombinant methods or utilize Peptide synthesizer synthetic.With paddy rice K ⁺/ Na ⁺The unmodified form bonded antibody of transporter gene product can come immune animal and produces with the gene product of producing in the prokaryotic cell prokaryocyte (for example E.Coli); With posttranslational modification form bonded antibody (as the albumen or the polypeptide of glycosylation or phosphorylation), can come immune animal and obtain with the gene product that produces in the eukaryotic cell (for example yeast or insect cell).Anti-paddy rice K ⁺/ Na ⁺The antibody of translocator can be used for the paddy rice K in the test sample ⁺/ Na ⁺Translocator.

The invention still further relates to quantitative and detection and localization paddy rice K ⁺/ Na ⁺The testing method of translocator level.These tests are known in the art, and comprise that FISH measures and radioimmunoassay.The paddy rice K that is detected in the test ⁺/ Na ⁺The translocator level can be used for explaining paddy rice K ⁺/ Na ⁺Translocator is importance aspect salt tolerance.

Whether there is paddy rice K in a kind of test sample ⁺/ Na ⁺The method of translocator is to utilize paddy rice K ⁺/ Na ⁺The specific antibody of translocator detects, and it comprises: with sample and paddy rice K ⁺/ Na ⁺The contact of translocator specific antibody; Observe whether form antibody complex, formed antibody complex and just represented to exist in the sample paddy rice K ⁺/ Na ⁺Translocator.

Part or all of polynucleotide of the present invention can be used as probe stationary on microarray (microarray) or DNA chip (being called " gene chip " again), is used for analyzing the differential expression analysis of tissue gene.Use paddy rice K ⁺/ Na ⁺The special primer of translocator carries out RNA-polymerase chain reaction (RT-PCR) amplification in vitro also can detect paddy rice K ⁺/ Na ⁺The transcription product of translocator.

In an example of the present invention, a kind of isolating polynucleotide are provided, its coding has the polypeptide of aminoacid sequence shown in the SEQ IDNO:2.Polynucleotide of the present invention are isolated from rice cDNA library, and its sequence is shown in SEQ ID NO:1, and the polynucleotide sequence total length that it comprises is 1665 bases, and its open reading frame is positioned at the 1-1662 position, and the coding total length is 554 amino acid whose paddy rice K ⁺/ Na ⁺Translocator (SEQ ID NO:2), it is a kind of 10 membranins of striding the film district that have.

Paddy rice K ⁺/ Na ⁺Translocator has clear and definite controlling plant K ⁺/ Na ⁺The function of transhipment.Therefore, paddy rice K ⁺/ Na ⁺The translocator for a change salt tolerance of plant provides new approach, thereby has great application prospect.Can import K ⁺/ Na ⁺Transporter gene can change the salt tolerance of existing good variety of crops, obtains the farm crop such as wheat, paddy rice of high-salt tolerance or other kinds such as woods grass, fruit tree, flowers and trees etc., solves the practical problems that agroforestry exist in producing.

Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to people such as normal condition such as Sambrook, molecular cloning: laboratory manual (New York:ColdSpring Harbor Laboratory Press, 1989) or molecular biology of plants-laboratory manual (PlantMolecular Biology-A Laboratory Mannual, Melody S.Clark compiles, Springer-verlag Berlin Heidelberg, 1997) condition described in, or the condition of advising according to manufacturer.

Embodiment 1

Paddy rice K ⁺/ Na ⁺The assignment of genes gene mapping of transporter gene and linkage analysis

Genetic group and molecule marker that utilization makes up with height salt water resistance rice varieties " Nona Bokra " (can available from the variety resources of rice storehouse of International Rice Research Institute etc.) and sense salt rice varieties " light more " (can available from the variety resources of rice storehouse from the International Rice Research Institute etc.) are to salt stress control paddy rice K down ⁺The following genome location of carrying out of the gene of ionic absorption.Press Haruhhina Y, etal, Genetics, 148,479-497, the method described in 1998 at first makes up a F2 who covers rice genome for the molecule marker linkage map, utilizes the corresponding F3 strain mensuration paddy rice K of system ⁺Ionic concn is carried out full genome scanning by the interval graphing method of routine then, the location genes involved.Found that paddy rice K under the control salt stress ⁺The major gene SKc1 of ionic absorption is positioned on No. 1 karyomit(e).

Further by following high for backcrossing analytical procedure: soon height salt tolerant kind " Nona Bokra " is hybridized acquisition F1 with sense salt kind " light more ", carry out backcrossing for 3-4 time as recurrent parent and F1 with sense salt kind " light more " again, obtain only to carry between the chromosomal region of height salt tolerant kind SKc1 gene and be the large-scale groups of heterozygosis separate condition, under the genetic background unanimity, utilize the known molecular mark that this gene is carried out the pinpoint accuracy linkage analysis.The result shows that this gene is positioned on the pac clone.

Embodiment 2

Paddy rice K ⁺/ Na ⁺The clone of transporter gene

(a) genome sequence

On the basis of pinpoint accuracy linkage analysis, from height salt water resistance rice varieties " NonaBokra " genome, be cloned into the SKc1 gene in order to following method.(as CAPS, SSR STS) screens exchange individuality in the extensive genetic group (about 7000 strains), measures the individual K of these exchanges to design highdensity molecule marker according to the base sequence of rice genome ⁺Concentration with this gene locking, is cloned into the SKc1 gene by round pcr (primer corresponds respectively to SEQ ID NO:3 1-28 position, and the 3969-3999 position) then from height salt water resistance rice varieties " Nona Bokra " genome.Genomic fragment length is 3.999Kb (SEQID NO:3), includes 3 exons (exon) and 2 introns (intron).Wherein exons 1 is positioned at 1-1238, and exon 2 is positioned at 2888-3115, and exon 3 is positioned at 3801-3999, and introne 1 is positioned at 1239-2887, and intron 2 is positioned at 3116-3800.

(b) cDNA sequence

Make up the cDNA library of height salt water resistance rice varieties Nona Bokra with ordinary method.With the cDNA library is template, with PCR method (forward primer 5 '-ATGAGTTCTCTGGATGCCAC-3 ' (SEQ ID NO:4); Reverse primer 5 '-TTATTCTATCTTCCATGCCTGACC-3 ' (SEQ ID NO:5)), obtain amplified production, be building up on the T carrier (Promega company) and check order, thereby obtain the total length ORF (openreading-frame) (SEQ ID NO:1) of SKc1 gene, its base length is 1.665Kb, 554 amino acid (SEQ ID NO:2) of encoding, the protein product molecular weight is estimated as 60.1KD, infers that it is to have 10 membranins of striding the film district.

Comparison shows that (Fig. 1) with other proteic aminoacid sequences, similarity (Similarity) and the consistence (Identity) of itself and barley gene TaHKT1 are respectively 52.3% and 43.7%; Be respectively 56.1% and 47.9% with similarity and the consistence of arabidopsis gene AtHKT1; Be respectively 54.4% and 43.7% with similarity and the consistence of paddy gene OsHKT1; With similarity and the consistence of another gene of paddy rice OsHKT4 be respectively 48.3% and 41.2%.This prompting SKc1 is K ⁺/ Na ⁺Transporter gene is a new gene member of HKT gene family.

Embodiment 3

The RT-PCR method obtains paddy rice K ⁺/ Na ⁺The encoding sequence of translocator

In order to verify the exactness of sequence,, design and synthesized following primer according to the sequence of SEQ ID NO:1:

Forward primer 5 '-ATGAGTTCTCTGGATGCCAC-3 ' (SEQ ID NO:4)

Reverse primer 5 '-TTATTCTATCTTCCATGCCTGACC-3 ' (SEQ ID NO:5).

Being template with the extractive Nona Bokra of ordinary method paddy rice (available from variety resources of rice storehouse, International Rice Research Institute) Poly A+RNA, carry out the RT-PCR amplification, amplification condition be 94 ℃ 5 minutes, 94 ℃ 30 seconds, 60 ℃ 30 seconds, 72 ℃ were carried out 35 circulations in 1 minute subsequently, extended 5 minutes with 72 ℃ at last.The electrophoresis detection pcr amplification product reclaims, be connected to adopt on the T-Easy carrier stop the thing fluorescent mark (Big-Dye, Perkin-Elmer, method USA), (Perkin-Elmer checks order on USA) at ABI 377 sequenators.Sequencing result has been verified paddy rice K ⁺/ Na ⁺The exactness of the complete encoding sequence of translocator (SEQ ID NO:2).

Embodiment 4

Paddy rice K ⁺/ Na ⁺The point mutation of translocator

To the K of height salt water resistance rice varieties " Nona Bokra " with sense salt rice varieties " light more " ⁺/ Na ⁺Transporter gene checks order once more and compares.Found that there are 6 base mutations in the SKc1 gene between height salt water resistance rice varieties " NonaBokra " and sense salt rice varieties " light more ", wherein the variation of the 418th, 551,994 and 1183 base causes 4 amino acid to replace (table 2).The variation in these sites may be to cause the discrepant reason of the gene function of this two kind.

Two kinds of K of table 2 ⁺/ Na ⁺The comparison of transporter gene

Position among the SEQ ID NO:1	Nona Bokra	Light more
			418	Nucleotide G (amino acid A)	Nucleotide C (amino acid P)
551	Nucleotide A (amino acid H)	Nucleotide G (amino acid R)
			994	Nucleotide G (amino acid D)	Nucleotide C (amino acid H)
1152	Nucleotide A (amino acid A)	Nucleotide G (amino acid A)
			1183	Nucleotide G (amino acid V)	Nucleotide C (amino acid L)
1305	Nucleotide C (amino acid G)	Nucleotide G (amino acid G)

Embodiment 5

Paddy rice K ⁺/ Na ⁺The functional analysis experiment of transporter gene

Height salt tolerant kind " Nona Bokra " obtains F1 with sense salt kind " light more " hybridization, carry out backcrossing for 4 times as recurrent parent and F1 with sense salt kind " light more " again and obtain BC4F1 (4 times backcross F1 for), in each backcross process, utilize molecule marker to select, obtain the plant that genetic background all reverts to recurrent parent " light more ".Utilize molecular marker-assisted selection method seed selection from BC4F2 (4 times backcross F2 generation) promptly to follow the tracks of in the genetic background of SKc1 genomic fragment (short-movie section) the importing recurrent parent " light more " of salt water resistance rice varieties highly at last by molecule marker to the near isogenic line SKc1-NIL (Nearly Isogenic Line) of SKc1.Utilize the base sequence of rice genome database to carry out predictive genes, the result is presented in this genomic fragment that is imported to be had only the SKc1 gene and not to have other gene about ion transporter gene, therefore has only the SKc1 gene to import in the sense salt kind " light more " from the genomic relevant ion transporter gene of height salt tolerant kind " Nona Bokra " near isogenic line SKc1-NIL.Utilize SKc1-NIL and recurrent parent " light more " to carry out (140mMNaCl) K under non-salt stress and the salt stress ⁺The mensuration of concentration.

The result shows, the K of SKc1-NIL and " light more " under non-salt stress ⁺Concentration does not have difference, and under salt stress the K of the overground part of SKc1-NIL ⁺Concentration is significantly higher than light (increasing by 27.28%) more, shows that salt tolerant kind SKc1 gene locus has obvious increase K ⁺The function and this gene that absorb also are subjected to inducing of salt stress; Measured Na in addition ⁺Concentration, result are presented at the leaf of SKc1-NIL, the Na in the stem ⁺Concentration significantly is lower than " light more ", and Na in the root of SKc1-NIL ⁺Concentration is higher than " light more " (Fig. 2).

This shows that the SKc1 gene of salt tolerant kind has the K that increases the paddy rice overground part under salt stress ⁺Absorb and reduction Na ⁺Function, thereby alleviate Na ⁺To the murder by poisoning of leaf, improve the salt tolerance of paddy rice.As seen this gene has using value in the breeding of farm crop resistance.This is at the relevant K of farm crop ⁺/ Na ⁺The new function of finding first in the transporter gene.

Embodiment 6

Paddy rice K ⁺/ Na ⁺The preparation of translocator

In this embodiment, be template with the amplified production of embodiment 3, use corresponding to 5 of this dna sequence dna ' and the PCR Oligonucleolide primers (SEQ ID NO.6 and 7) of 3 ' end increase, obtain cDNA as inserting fragment.

5 ' Oligonucleolide primers sequence is:

5 '-GCGGATCCATGAGTTCTCTGGATGCCAC-3 ' (SEQ ID NO.6 contains the restriction enzyme site of BamHI restriction enzyme);

3 ' end primer sequence is:

5 '-CGAAGCTTTTATTCTATCTTCCATGCCTGACC-3 ' (SEQ ID NO.7 contains the restriction enzyme site of HindIII restriction enzyme).

The restriction enzyme site of the restriction enzyme on the primer is corresponding to bacterial expression vector pQE-9 (Qiagen Inc., Chatsworth, CA) restriction enzyme digestion sites on, this plasmid vector coding antibiotics resistance (Ampr), a bacterium replication orgin (ori), an adjustable promotor/operon of IPTG-(P/O), a ribosome bind site (RBS), a 6-histidine mark thing (6-His) and restriction enzyme cloning site.

With BamHI and HindIII digestion pQE-9 carrier and insertion fragment, will insert fragment subsequently and be connected to the pQE-9 carrier and keep open reading frame initial at bacterium RBS.Transform available from Qiagen with connecting mixture subsequently, the E.coli bacterial strain of commodity M15/rep4 by name, M15/rep4 contains the plasmid pREP4 of multiple copied, and it is expressed the lacI repressor and carries kalamycin resistance (Kanr).Screen transformant containing on the LB culture dish of Amp and Kan, the extracting plasmid, the cDNA fragment of sequence verification SKc1 has correctly been inserted carrier.

Incubated overnight (O/N) contains the positive transformant clone of required construction in the LB liquid nutrient medium of adding Amp (100 μ g/ml) and Kan (25 μ g/ml).Spend the night (O/N) culture with 1: 100-1: 250 thinning ratio dilution, be inoculated into then in the large volume substratum, culturing cell grows to 600 optical density(OD) (OD ₆₀₀) when being 0.4-0.6, add IPTG (" isopropylthio-") to final concentration be 1mM.By making lacI repressor inactivation, IPTG induces startup P/O to cause gene expression dose to improve.Continued culturing cell 3-4 hour, centrifugal subsequently (6000 * g, 20 minutes).The ultrasonic degradation inclusion body, collecting cell also is dissolved in cell precipitation in the Guanidinium hydrochloride of 6M.After the clarification, by containing under the condition that 6-His marker albumen combines closely making, with nickel-chelate column chromatography purifying dissolved SKc1 from solution.With 6M Guanidinium hydrochloride (pH5.0) wash-out SKc1 from post.Available several method is the sex change protein precipitation from Guanidinium hydrochloride.Perhaps, use the dialysis step to remove Guanidinium hydrochloride, perhaps isolated purifying protein from nickel-chelate column.Protein behind the purifying is incorporated in second post, has the linear Guanidinium hydrochloride gradient of successively decreasing in this post.Protein denaturation when being attached to this post is used Guanidinium hydrochloride (pH5.0) wash-out subsequently.At last, soluble protein is dialysed with PBS, then protein is kept in the stock solution that final concentration is 10% (w/v) glycerine.

SDS-PAGE glue with 12% carries out electrophoresis, identifies that the molecular weight size of expressing protein is about 60KDa.

In addition, the amino acid of expressing proteic N end and each 10 amino acid length of C end is checked order, find consistent with the sequence of SEQ ID NO.2 with ordinary method.

Embodiment 7

Preparation antibody

The recombinant protein that obtains among the embodiment 6 is used for immune animal to produce antibody, and concrete grammar is as follows.Recombinant molecule is standby after separating with chromatography.Also available SDS-PAGE gel electrophoresis separates, electrophoretic band downcut from gel, and with isopyknic complete Freund ' s adjuvant emulsion.Albumen with 50-100 μ g/0.2ml emulsification carries out peritoneal injection to mouse.After 14 days,, mouse is carried out peritoneal injection with booster immunization with the dosage of 50-100 μ g/0.2ml with the same antigen of non-complete Freund ' s adjuvant emulsion.Carried out booster immunization one time every 14 days, carry out at least three times.The sero-fast specific reaction that obtains is active to be assessed in the ability of external precipitation SKc1 gene translation product with it.Found that antibody can precipitate with albumen of the present invention specifically.

Embodiment 8

Paddy rice SKc1 transgenosis

Present embodiment adopts binary vector pCAMBIA1300 commonly used, and (available from CAMBIA company, Canberra is Australia) as the paddy rice transgene carrier.A bacterium replication orgin of this vector encoded (ori), kalamycin resistance gene (Kanr), hygromycin gene (Hygr) and restriction enzyme cloning site (MCS).The genomic dna or the cDNA that can insert SKc1 at the restriction enzyme cloning site are built into the transgenosis plasmid.

(1) the transgenosis plasmid construction of band SKc1 genomic dna:

Utilize PCR method to divide 4 sections (Oligonucleolide primers is respectively SEQ ID NO.9 and 10,11 and 12,13 and 14,15 and 16) amplification as template with height salt tolerant kind " Nona Bokra " genomic dna.

SEQ ID NO.9：5’-AGCTGGGAGTTGGAGAAGGACT-3’

SEQ ID NO.10：5’-CGACGTGACAGATTTGGATTAC-3’

SEQ ID NO.11：5’-CCAAATCAAGGGCTCCAA-3’

SEQ ID NO.12：5’-ACTTCCTCTTCATCTCCC-3’

SEQ ID NO.13：5’-TGCTGGTGTTTGTCTTGGACAG-3’

SEQ ID NO.14：5’-ATATGGTCTTCGATGCTGTGGC-3’

SEQ ID NO.15：5’-GAGGGAGAAGAAGTGATTTT-3’

SEQ ID NO.16：5’-TGAATTATACTGCGTGAACC-3’

Connect into the genomic DNA fragment of 7.276Kb respectively with BstxI, NarI, KpnI restriction endonuclease, comprise the SKc1 gene coding region.This fragment also comprises 723 bases of 2554 bases and terminator codon TAA downstreams of translation initiation codon ATG upstream.This segmental 5 ' end connects behind EcoRI joint and is connected with the pCAMBIA1300 (available from CAMBIA company) of SmaI digestion through EcoRI.The connector transformed into escherichia coli is used bacterial strain DH5 α always, on the 2YT substratum that contains Kan (50 μ g/ml) and X-gal, screen transformant, select white colony and extract plasmid, insert carrier with EcoRI and the about 7.3Kb fragment of NotI enzymolysis checking, and whether correct with M13 universal primer order-checking check nucleotide sequence.

(2) the transgenosis plasmid construction of band SKc1 gene cDNA:

In this embodiment, amplified production with embodiment 3 is a template, use the PCR Oligonucleolide primers (SEQ ID NO.6 and 8) corresponding to 5 of this dna sequence dna ' and 3 ' end to increase, the cDNA fragment of acquisition is inserted pCAMBIA1300 and is gone up corresponding restriction enzyme cloning site.Insert the CaMV35S promotor between the restriction enzyme cloning site EcoRI of this plasmid and BamHI, inserted the termination signal sequence of NOS gene between XbaI and HindIII.

5 ' Oligonucleolide primers sequence is:

5 '-GCGGATCCATGAGTTCTCTGGATGCCAC-3 ' (SEQ ID NO.6 contains the restriction enzyme site of BamHI restriction enzyme);

3 ' end primer sequence is:

5 '-CGTCTAGATTATTCTATCTTCCATGCCTGACC-3 ' (SEQ ID NO.8 contains the restriction enzyme site of XbaI restriction enzyme).

With BamHI and XbaI digested cdna and carrier pCAMBIA1300, cDNA is connected between the termination signal sequence of CaMV35S promotor and NOS gene.Connector transformed into escherichia coli bacterial strain DH5 α, on the 2YT substratum that contains Kan (50 μ g/ml), screen transformant, select single bacterium colony and extract plasmid, pick out the clone that the 1.7Kb fragment of having an appointment is inserted with BamHI and XbaI enzymolysis, and whether correct with M13 universal primer order-checking check nucleotide sequence.

(3) SKc1 gene transformation paddy rice: above-mentioned two kinds of recombinant plasmids import agrobacterium strains EHA105 commonly used by freeze-thaw method.Per 200 μ l EHA105 competent cells add 0.5-1 μ g (about 10 μ l) plasmid DNA mixing, successively on ice, respectively placed 5 minutes in liquid nitrogen and 37 ℃ of water-baths; Be diluted to 1ml with fresh YEB liquid nutrient medium, cultivated 2-4 hour in 28 ℃ of joltings; Get 200 μ l and coat on the YEB flat board that contains microbiotic Kan (50 μ g/ml), cultivated 2-3 days for 28 ℃.The bacterium colony that grows is drawn 3 times continuously containing stroke single bacterium on the antibiotic YEB flat board.Method with reference to (1994) such as Hiei, the single colony inoculation of picking Agrobacterium contains the antibiotic YEB liquid nutrient medium in 28 ℃ of jolting overnight incubation to 3ml from the YEB flat board, contained in the antibiotic AB liquid nutrient medium by the 1% inoculum size 50ml that transfers in the 2nd day, 200rpm continues jolting and is cultured to OD ₆₀₀When being 0.6 to 0.8 left and right sides, fresh Agrobacterium bacterium liquid in centrifugal 5 minutes of 5000rpm, 4 ℃, is collected and is resuspended in the AAM liquid nutrient medium of 1/3 volume, this bacterium liquid promptly can be used for the various acceptor materials of rice transformation.

Present embodiment adopts the rataria callus of spending 11 (sense salt kind is available from Chinese Academy of Agricultural Sciences's crop varieties resources banks) in the conventional conversion method for agrobacterium rice transformation.Get pollination back 12-15 days in spend 11 immature seeds through 70% alcohol immersion after 1 minute, with 1.5%NaClO solution disinfection 90 minutes, aseptic water washing 4-5 time, then with scalper with take the photograph son and choose rataria and be inoculated in N6D ₂Evoked callus on the substratum is cultivated under 26 ± 1 ℃, lucifuge condition, can be used for after 4 days transforming.The rataria callus is soaked in the fresh AAM Agrobacterium bacterium liquid and shakes frequently, after 20 minutes rice material is shifted out, on aseptic filter paper, inhale and remove too much bacterium liquid, transfer to N6D immediately ₂On the C substratum, cultivated altogether 3 days in 26 ℃.When cultivating altogether, adding Syringylethanone as Agrobacterium Vir gene activation thing in the culture medium altogether, working concentration is 100 μ mol/L.After 3 days, take out callus, cut plumule and change over to and select substratum N6D from being total to culture medium ₂S1 (Hyg 25mg/l) selects to cultivate.Forward resistant calli to N6D2S after 7-12 days ₂(Hyg 50mg/l) selects to continue on the substratum screening.Eugonic resistant calli is transferred on the pre-differentiation substratum and is cultivated about a week after 10-12 days, moves to differentiation (12 hours illumination/skies) on the division culture medium again.The regenerated seedling is at 1/2MS ₀Strong plantlets and rootage on the H substratum moves into the cultivation of phytotron basin soil subsequently.Extract the total DNA of blade behind the regeneration plant transplant survival that obtains, identify transformed plant through PCR or Southern hybridization.Carry out K under the salt stress with transgenosis T1 generation ⁺, Na ⁺Ionic concn is measured.

The result shows that the salt tolerance of transgenic paddy rice increases, this checking SKc1 gene function.

All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.

Sequence table

＜110〉Shanghai Inst. of Life Science, CAS

＜120〉paddy rice potassium, sodium ion transporter gene and application thereof

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＜213〉paddy rice (Oryza sativa)

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gtcgtcaacc ctaccaccac tgcgacgacg cacgacgagg tagagctagg gttaggacgt 540

cggaacaagc acggctgcac ctgcactact actcacacgt cgtcgtcatc atcggcatcg 600

aagacgacga cgacgaggct actgatgttc gtggtgatgg ggtaccacgc ggtggtgcac 660

gtcgccgggt acacggccat cgtcgtgtac ctcagcgccg tcggcggcgc gggggcggtg 720

gtcgccggga aggggatcag cgcgcacacg ttcgccatct tcaccgtcgt ctcgacgttc 780

gccaactgcg ggttcgtgcc gacgaacgaa gggatggtgt cgttcaggtc gttcccgggg 840

ctcctcctcc tcgtcatgcc gcacgtcctc ctcgggaaca cgctcttccc ggtcttcctc 900

aggctggcca tcgccgcgct cgagagggtc accgggtggc cggagctcgg cgagctcctg 960

atccggcggc ggaggggcgg cggcgagggc tacgaccacc tgttgccgag ctcgcgcacg 1020

cggttcctgg ccctcaccgt ggccgtgctc gtggtggcgc agctggcgct cttctgcgcc 1080

atggagtggg gctccgacgg gctgcggggg ctcaccgccg gccagaagct cgtcggcgcg 1140

ctcttcatgg cagtcaactc gaggcactcc ggtgagatgg tggtcgacct ctccaccgtg 1200

tcgtcggccg tcgtcgtgct ctacgtggtg atgatgtatg tatatttgtc ctcctcaatt 1260

attatatttt tgacaggccc tccttaatat attaattact atcacatgca tgtgtgcaca 1320

cgtaaaaact aaataaatta ccatatcata catgtgggta cttaatttag tactctggcc 1380

tgggacatat tttaaatcct attggacatc atcctttcaa tttttttttt gaaacaaagt 1440

aaaaacgcag gcgcttataa cgcgtgcaca ctcaccctta tgaatacaaa tcttacccct 1500

ataagtacct tcgaaagact gagctgatat atcttaagat tgatgaagtc atcacgggtg 1560

tctcactgtc gacaagtaca tcactaccta ccactaaaag aataattagc cataaatata 1620

agcacccgtg tcaaatttat tatttgaatc tttcattcta attatatgcg atcaaaatag 1680

ttatgaaaaa ttctacttta tatgggggtg cagggtctcg aggcatctaa atagttatga 1740

aaaaatctat aaaaatacaa gagatatatg gtcttcgatg ctgtggcaca agtggactag 1800

tggttgtcat tttacgcgct caaaatgtaa ccgaacgagg cgccgtcatg gatcactatc 1860

aaaagcagct tagtaccgaa ttaatatatg tggctacaaa tgctaatcag ctaaatgata 1920

ggaaagattg attgctactg atagacatgg attgacgctt tcaggttgtt ggagcccttg 1980

atttggcact ttcacgacga taatccgcaa ggataattct ccaagtcata ttttgcaaca 2040

aaatacaact acatctagtt taatttcctc taagttagta agttgtgact gcaaattaac 2100

aagtgtattg atgtccgaaa cctagctaga tgaaggttat atacattgga tagggatcga 2160

aaaaaaaata gacagaaaca gagaacgata agtcaattgc aagcacatac tctctccata 2220

ctcataaagg aagtcattta agacaatgtt taagtcaaac cttgggatta taaatcataa 2280

ataactctca agttgttgag tttgaaaatg taaaaattat atgaataaat ttgtcttgaa 2340

aaatactttc ataaaagtat acatatatca cttttcaatg aatattttta tagaaagaag 2400

aagtcaaagt tgtattttgg agaccgtgtc gatgtcctaa acgacttcct ttatgagtat 2460

ggagggagta tatatactct gtacttaagt atacgataat gttatctttg ttcagtgaaa 2520

cttttggaac tttgattgtt actagctatc aaaattttta gtttgagaat ataaaaatta 2580

ttaatattat aattttgtta gatattatcg ttatgtttta ataaaacaac ggtggtcaaa 2640

gtagtacaca ccaaacaccg aatgaagtca acatcggtgt gtgtatatat atacgcggag 2700

gtagtacatg atacaacttt tgaattaaac tcccgaaaaa catatgttct catgcatgct 2760

tttgtataca gatatgatat agctctaaat taataacttg catgacggaa attgagcata 2820

tttactacac tgaattatac tgcgtgaacc ttaatttgct tatatattct catgcttgct 2880

gcaggtacct gccaccttac accactttcg tacctgtcca agacaaacac cagcaaacgg 2940

gagcacagtc cggccaggag ggcagcagca gcagcagcat atggcagaag ctgctcatgt 3000

cgccgctctc gtgcctagcc atcttcatcg tcgtcatctg catcacggag cggcggcaaa 3060

tcgccgacga ccccatcaac tacagcgtcc tcaacatcgt cgtcgaggtt atcaggtacg 3120

catagtggtg gtctaacctt aattttaagt gctggcaagt gccaaaaaat tactctctcc 3180

atctcttaaa tatttgacgc cgttgacttt ttaaaacatt ttaaccgttc gtctttttgt 3240

cttatcaaaa atttttaagt aattattaat tctttttcta tcatttattt attactaaat 3300

atatttttat gtatacatat agttttacat atttctcaaa agtttttaat aagacgaacg 3360

gtcgaacatg tttaaaaaag tcaacaacgt caaatattta gggacagaga gtatatttcc 3420

cccttgaggg tacatacatt tctcctcgtt tttatatgtc atcccaaaaa ttacaaaaga 3480

aaatctaaaa gttattgata aaatggacat gtgatatatt actacactaa tatatacacg 3540

ttcaaatata acttgaacac atgttagaaa aacacaataa atttaactat aaatggcatg 3600

ttcaattcgc aattaaattt aatattttat ttctgtttga acaaattgaa tatcatatat 3660

ttaaaaagag attaattaat gattttttaa acgaatggat aaaataagag aaagtgagga 3720

aaatccacat ccacggagat aatatatata actagaacca tcgtgtaaac taactatatg 3780

ttacatactc ctggatgcag tgcgtatggc aatgtggggt tcagcacggg gtacagctgc 3840

gcgaggcagg tgaggcccga cggcagctgc agagacctgt gggttggctt ctcagggaag 3900

tggagcaaac aagggaagct cactctcatg gccgtcatgt tctacggcag gctcaagaag 3960

ttcagcctgc acggtggtca ggcatggaag atagaataa 3999

<210>4

<211>20

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>(1)..(20)

＜223〉primer

<400>4

atgagttctc tggatgccac 20

<210>5

<211>24

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>(1)..(24)

＜223〉primer

<400>5

ttattctatc ttccatgcct gacc 24

<210>6

<211>28

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>(1)..(28)

＜223〉primer

<400>6

gcggatccat gagttctctg gatgccac 28

<210>7

<211>32

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>(1)..(32)

＜223〉primer

<400>7

cgaagctttt attctatctt ccatgcctga cc 32

<210>8

<211>32

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>(1)..(32)

＜223〉primer

<400>8

cgtctagatt attctatctt ccatgcctga cc 32

<210>9

<211>22

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>(1)..(22)

＜223〉primer

<400>9

agctgggagt tggagaagga ct 22

<210>10

<211>22

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>(1)..(22)

＜223〉primer

<400>10

cgacgtgaca gatttggatt ac 22

<210>11

<211>18

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>(1)..(18)

＜223〉primer

<400>11

ccaaatcaag ggctccaa 18

<210>12

<211>18

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>(1)..(18)

＜223〉primer

<400>12

acttcctctt catctccc 18

<210>13

<211>22

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>(1)..(22)

＜223〉primer

<400>13

tgctggtgtt tgtcttggac ag 22

<210>14

<211>22

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>(1)..(22)

＜223〉primer

<400>14

atatggtctt cgatgctgtg gc 22

<210>15

<211>20

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>(1)..(20)

＜223〉primer

<400>15

gagggagaag aagtgat ttt 20

<210>16

<211>20

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>(1)..(20)

＜223〉primer

<400>16

tgaattatac tgcgtgaacc 20

Claims (10)

1. a peptide species, described polypeptide is isolating paddy rice K ⁺/ Na ⁺Translocator is characterized in that, described polypeptide is selected from down group:

(a) has the polypeptide of SEQ ID NO:2 aminoacid sequence; Or

(b) replacement, disappearance or the interpolation of SEQ ID NO:2 aminoacid sequence through one or more amino-acid residues formed, and have the plant of promotion K ⁺/ Na ⁺Transport function by (a) polypeptides derived.

2. polypeptide as claimed in claim 1 is characterized in that this amino acid sequence of polypeptide is shown in SEQ IDNO:2.

3. isolating polynucleotide is characterized in that, the described polypeptide of described polynucleotide encoding claim 1.

4. polynucleotide as claimed in claim 3 is characterized in that this polynucleotide encoding has the polypeptide of aminoacid sequence shown in the SEQ IDNO:2.

5. polynucleotide as claimed in claim 3 is characterized in that, the sequence of these polynucleotide is selected from down a kind of of group:

(a) has the nucleotide sequence of 1-1665 position among the SEQ ID NO:1;

(b) has the nucleotide sequence of 1-1662 position among the SEQ ID NO:1;

(c) has the nucleotide sequence of 1-3999 position among the SEQ ID NO:3.

6. a carrier is characterized in that, it contains the described polynucleotide of claim 3.

7. a genetically engineered host cell is characterized in that, it contains and is integrated with the described polynucleotide of claim 3 in described carrier of claim 6 or the genome.

8. paddy rice K ⁺/ Na ⁺The preparation method of translocator is characterized in that, this method comprises:

(a) under conditions suitable for the expression, cultivate the described host cell of claim 7;

(b) from culture, isolate paddy rice K ⁺/ Na ⁺Translocator.

9. method that improves plant is characterized in that it comprises step:

(1) provide the Agrobacterium of carrying expression vector, described expression vector contains paddy rice K ⁺/ Na ⁺The encoding sequence of translocator, described paddy rice K ⁺/ Na ⁺Translocator is selected from down group:

(a) has the polypeptide of SEQ ID NO:2 aminoacid sequence;

(b) replacement, disappearance or the interpolation of SEQ ID NO:2 aminoacid sequence through one or more amino-acid residues formed, and have the plant of promotion K ⁺/ Na ⁺Transport function by (a) polypeptides derived;

(2) vegetable cell or tissue or organ are contacted with Agrobacterium in the step (1), thereby make paddy rice K ⁺/ Na ⁺Translocator dna encoding sequence changes vegetable cell over to, and is incorporated on the karyomit(e) of vegetable cell;

(3) select and change paddy rice K over to ⁺/ Na ⁺The vegetable cell of translocator dna encoding sequence or tissue or organ;

(4) vegetable cell in the step (3) or tissue or neomorph are become plant.

10. method as claimed in claim 9 is characterized in that, the salt tolerance of described method improvement plant.

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