CN1238374C

CN1238374C - Na+/H+ retrograde transport protein of New Zedland spinach, its coding gene and application

Info

Publication number: CN1238374C
Application number: CN 03109436
Authority: CN
Inventors: 李银心; 吕慧颖
Original assignee: Institute of Botany of CAS
Current assignee: Institute of Botany of CAS
Priority date: 2003-04-09
Filing date: 2003-04-09
Publication date: 2006-01-25
Anticipated expiration: 2023-04-09
Also published as: CN1535982A

Abstract

The present invention discloses a Na+/H+ retrograde transport protein of New Zealand spinach, a coding gene and application thereof. The provided Na+/H+ retrograde transport protein of New Zealand spinach comes from tetragonia tetragonioides O, with the name of TtNHX1. The Na+/H+ retrograde transport protein of New Zealand spinach is protein with an amino acid residue sequence of a sequence 2 in a sequence list or is the protein derived from the sequence 2, wherein the amino acid residue sequence of the sequence 2 is replaced or lost or added by one or a plurality of amino acid residues and the protein has the same activity as the activity of the amino acid residue sequence of the sequence 2. The coding gene TtNHX1 of the Na+/H+ retrograde transport protein of New Zealand spinach is one of the following nucleotide sequences: 1), SEQ ID No: 1 in the sequence list; 2), polynucleotide of the protein sequence of SEQ ID No: 2 in a coding sequence list; 3), a DNA sequence which has more than 90% of isogenous performance with the DNA sequence limited by the SEQ ID No: 1 in the sequence list and can code the protein with identical functions. The gene has important significance to the cultivation of salt resistance plant varieties and the enhancement of crop yield.

Description

The Na of New Zealand spinach +/ H +Its encoding gene of Reverse transship albumens And and application

Technical field

The present invention relates to a kind of translocator and encoding gene and application in the plant genetic engineering field, particularly relate to the Na of New Zealand spinach ⁺/ H ⁺Its encoding gene of Reverse transship albumens And and application.

Background technology

Salt damage is the serious limiting factor of crop yield.Understand the anti-salt mechanism of plant, clone's salt resistant gene is also transferred in the salt-sensitive farm crop, cultivates the genetically modified crops new variety of anti-salt, for China that more than 500,000,000 mu of salt wasteland is arranged, has crucial meaning.

Plant has formed a series of adaptation mechanism in order to resist salt stress, mainly is to realize osmotic equilibrium by the micromolecular non-toxic organic solute of accumulation and ion separating in vacuole in tenuigenin.The higher plant cell vacuole accounts for 95% of complete expansion cell cumulative volume, Na for plant ⁺Be the cell needed basic nutrition composition of growing, Na ⁺Separating be the necessary guarantee that it is survived in salt environment.Na ⁺/ H ⁺Reverse transport protein (antiporter) is a kind of Na ⁺The antiport body, distribution is all arranged, the Na on the vacuole skin on cytoplasmic membrane and vacuole skin ⁺/ H ⁺Antiporter is with the Na in the tenuigenin ⁺Against Na ⁺Concentration gradient is transported in the vacuole separating and concentrates, the Na on the cytoplasmic membrane ⁺/ H ⁺Antiporter is with the Na in the tenuigenin ⁺The reverse outer high Na of born of the same parents that is transported to ⁺In the environment, thereby reduced the murder by poisoning of organoid in the sodium ion pair cell matter, played an important role for the osmotic equilibrium of keeping cell.

Na in the plant ⁺/ H ⁺Antiporter finds it is on the barley plasma membrane the earliest, and after this research report is all arranged on plasma membrane and vacuole skin, finds Na in a large amount of halophytess and non-halophytes up till now ⁺/ H ⁺The antiporter vigor.Separated Na from Arabidopis thaliana (AtNHX1), paddy rice (OsNHX1), northern saltbush (AgNHX1), ice leaf Herba Portulacae Grandiflorae (McNHX1) ⁺/ H ⁺The antiporter gene proves that this expression of gene product has Na ⁺/ H ⁺The function of exchange.Though these genes overwhelming majority is isolating from the glycophyte with low reverse transportation vigor, former study shows that halophytes generally has in vacuole efficiently separating Na ⁺Mechanism.To halophytes Na ⁺/ H ⁺The antiporter gene is only northern saltbush with alkali is fluffy that research was arranged, and the work among the latter is not seen formally and delivered.

Salt tolerance belongs to complex character, and this viewpoint is also supported in the existence of a large amount of salt stress reacting phase correlation genes.But term single gene generally can not significantly improve the salt tolerant level of crop, needs the synergy plant of a plurality of resistant gene of salt just can have high-salt tolerance.Yet the Arabidopis thaliana plant that changes the AtNhx1 gene can grow by normal growth in 200mM NaCl.The AtNhx1 gene of Arabidopis thaliana is changed in the tomato over to overexpression vacuole Na ⁺/ H ⁺The transgenic Fructus Lycopersici esculenti of antiporter can normal growth under 200mM NaCl coerces, bloom and solid.Although Na in the blade ⁺Concentration height, but Na in the tamato fruit ⁺Concentration is very low.Illustrating changes Na ⁺/ H ⁺The antiporter single-gene can obviously improve plant salt endurance, simultaneously, has the specificity that organ is expressed, and has provided powerful support for Na ⁺/ H ⁺The vital role that antiporter is had aspect plant salt endurance.

New Zealand spinach is annual meat herbaceous plant, and stronger salt tolerance is arranged, and originates in ground such as Australia, South East Asia and Chile, and now also there is cultivation in China, and it is edible that its young stem and leaf can be done vegetables.But the salt tolerant mechanism to New Zealand spinach is not studied from molecular level as yet.

Summary of the invention

The purpose of this invention is to provide New Zealand spinach Na ⁺/ H ⁺Its encoding gene of Reverse transship albumens And.

New Zealand spinach Na provided by the present invention ⁺/ H ⁺Reverse transport protein derives from New Zealand spinach (Tetragoniatetragonioides O.), name is called TtNHX1, be protein, or the amino acid residue sequence of sequence 2 is passed through replacement, disappearance or the interpolation of one or several amino-acid residue and has identical active by sequence 2 deutero-protein with the amino acid residue sequence of sequence 2 with sequence 2 amino acid residue sequences in the sequence table.

The protein that sequence 2 amino acid residue sequences are made up of 554 amino-acid residues in the sequence table.

According to Na among the GenBank ⁺/ H ⁺2 degenerated primerses, P1:5 '-ATTGG (T/A) GCAAT (A/C) TT (C/T) GCTGC-3 ' have been synthesized in the conservative aminoacid sequence design of reverse transport protein;

P2:5 '-TCTCAAT (A/g) TCCA (A/g) is gCATCC-3 ' (T/g/A), clones Na by RT-PCR and RACE method from the halophytes New Zealand spinach ⁺/ H ⁺The reverse transport protein gene.

New Zealand spinach Na ⁺/ H ⁺The encoding gene TtNHX1 of reverse transport protein is one of following nucleotide sequences:

1) the SEQ ID № in the sequence table: 1;

2) SEQ ID № in the code sequence tabulation: the polynucleotide of 2 protein sequences;

3) with sequence table in SEQ ID №: 1 dna sequence dna that limits has 90% above homology, and the identical function protein DNA sequence of encoding.

The dna sequence dna of sequence 1 is by 2199bp based composition in the sequence table, and the reading frame of this gene is to hold the 92nd to the 1756th bit base from 5 '.

New Zealand spinach Na ⁺/ H ⁺The clone of reverse transport protein gene helps the further 26S Proteasome Structure and Function feature of studying reverse transport protein on molecular level.To halophytes and non-halophytes, plasma membrane type and vacuole type Na ⁺/ H ⁺The reverse transport protein gene compares analysis, further the anti-salt mechanism of clear and definite plant on cell and molecular level.Utilize any carrier that can guide foreign gene in plant, to express, with coding New Zealand spinach Na provided by the present invention ⁺/ H ⁺The gene transfered plant cell of reverse transport protein TtNHX1, can obtain high-salt stress tolerance enhanced transgenic cell line and transfer-gen plant, to cultivate the genetically modified crops new variety of anti-salt, make its normal growth on the ground at saline soil, for the China that has more than 500,000,000 mu of salt wasteland, the economic benefit of this measure is very considerable.And the development of the future of agriculture is faced with increased population, the challenge that reduce in the arable land, and making full use of the saltings will play a great role.

Vacuole type reverse transport proteins such as New Zealand spinach TtNHX1 and AtNHX1, AgNHX1 have high homology, and Phylogenetic Analysis also shows TtNHX1 and vacuole type Na ⁺/ H ⁺The reverse transport protein homology is higher than the plasma membrane type.Therefore the function of TtNHX1 may be to stride vacuole skin transportation Na ⁺, it is accumulated in vacuole.In the Mammals the phonetic binding site of ammonia chloropyrazine identified into: ¹⁶³DVFFLFLLPPI ¹⁷³, the phonetic inhibition of ammonia chloropyrazine Na ⁺/ H ⁺The vigor of reverse transport protein.This regional sequence is in the New Zealand spinach ⁸⁵LFFIYLLPPI ⁹⁴, with AtNHX1, therefore OsNHX1 and NHX height homology also is convenient to better the different of relatively halophytes and glycophyte reverse transport protein for the research of New Zealand spinach TtNHX1.

Description of drawings

Fig. 1 is Na ⁺/ H ⁺Reverse transport protein cDNA clone

Fig. 2 is a Southern blot analytical results

Fig. 3 is a Northern blot analytical results

Fig. 4 is New Zealand spinach (TtNHX1) hydrophobicity analysis

Fig. 5 is plant Na ⁺/ H ⁺The reverse transport protein aminoacid sequence

Fig. 6 is Na ⁺/ H ⁺The phylogenetic tree analysis of reverse transport protein

Fig. 7 induces the expression of TtNHX1 in the different time New Zealand spinach for 200mM NaCl

Embodiment

The Na of embodiment 1, New Zealand spinach ⁺/ H ⁺The clone of reverse transport protein encoding gene

Concrete experimental procedure is as follows:

(1) RNA and DNA's separates

Extract RNA with Trizol test kit (GIBCO-BRL) vermiculite from New Zealand spinach (Tetragonia tetragonioides O.) seedling (sowing is grown under the greenhouse experiment), the DNase (TaKara) with no RNase removes DNA residual among the RNA subsequently.

DNA extraction adopts the CTAB method.

(2) New Zealand spinach Na ⁺/ H ⁺The segmental separation of reverse transport protein cDNA

Na in, the citrus fluffy according to northern saltbush, alkali ⁺/ H ⁺The synthetic degenerated primers of aminoacid sequence that reverse transport protein is conservative,

P1：5′-ATTGG(T/A)GCAAT(A/C)TT(C/T)GCTGC-3′；

P2：5′-TCTCAAT(A/g)TCCA(A/g)(T/g/A)gCATCC-3′。

In order to separate the cDNA fragment, with the synthetic first chain cDNA of reverse transcription test kit (Promega).The RT-PCR reaction mixture is 25 μ l, contains 2 μ l cDNA templates, 0.8mmol/L dNTPs, every primer of 0.6 μ mol/L, 1 * PCR damping fluid, 1U Taq DNA polymerase (Promega).The PCR response procedures: 95 ℃, 4min; 95 ℃ of 30sec, 50 ℃ of 30sec, 72 ℃ of 1min, totally 36 circulations; 72 ℃ of 10min.Obtain the cDNA fragment (Fig. 1 (b)) of an about 500bp.It is connected with PGEM T-easy carrier (Promega), and transformed into escherichia coli DH5 α utilizes blue hickie screening, the single bacterium colony of picking white, and the upgrading grain, Not I digested plasmid is identified the positive colony that is inserted with the big or small fragment of expection (500bp), checks order.Sequencing result shows, has obtained the cDNA segment of 535bp, with blast comparison New Zealand spinach cDNA fragment, finds this cDNA fragment and the Na that has cloned ⁺/ H ⁺The reverse transport protein gene has higher homology, illustrates that the New Zealand spinach cDNA segment that obtains is the fragment of Na+/H+ reverse transport protein gene.

(3) New Zealand spinach Na ⁺/ H ⁺The separation of reverse transport protein cDNA end

According to the Na that has cloned ⁺/ H ⁺Reverse transport protein cDNA fragments sequence synthetic gene special primer

3-GSP1：5′-CATGGCATAACGTGACTGAGAGC-3′；

3-GSP2：5′-TGAGAGCTCAAGAGTAACCACCAAG-3′；

5-GSP1：5′-GGCATTGAAAAGCACCACTG-3′；

5-GSP2：5′-CTACACCTTCCCCAAACACCAGACT-3′；

5-GSP3：5′-ACTGTATAGCAAAGGTGTCTCGTCC-3′。

Separate Na by the terminal rapid amplifying of cDNA (GIBCO-BRL) ⁺/ H ⁺Terminal and the 5 ' end of 3 of reverse transport protein cDNA '.With gene specific primer 3-GSP1,3-GSP2 and 3 ' RACE, 5-GSP1,5-GSP2,5-GSP3 and 5 ' RACE test kit separate cDNA3 ' end and 5 ' end.CDNA5 ' end sequence (Fig. 1 (a)) and 3 ' end sequence (Fig. 1 (c)) have been cloned with 5 ' RACE and 3 ' RACE method, sequencing result shows 5 ' and the length of 3 ' end fragment be respectively 628bp and 1221bp, they and middle 535bp cDNA fragment associated end have the overlap of 81bp and 104bp respectively, with these 3 fragment assemblies, obtain a total length 2199bp New Zealand spinach Na ⁺/ H ⁺The full-length cDNA of reverse transport protein gene (Fig. 1 (d)) is the sequence in the sequence table 1.

Embodiment 2, New Zealand spinach Na ⁺/ H ⁺Reverse transport protein cDNA complete sequence analysis

By the sequences Design special primer of splicing, with conventional RT-PCR method clone New Zealand spinach Na ⁺/ H ⁺The reverse transport protein full-length cDNA is accurately to its sequence verification splicing.This cDNA comprise 1665bp opening code-reading frame (Open ReadingFram, ORF), 5 ' non-translational region of 91 bases (Non Translated Region, NTR), the poly-A tail of 3 ' non-translational region of 412 bases and 31 bases.In 5 ' non-translational region+59 positions a terminator codon (TAG) is arranged, and match with the reading frame of coding region, there is not other more preceding initiator codon in this explanation, shows that this cDNA is a total length, with its called after TtNHX1.554 amino acid whose polypeptide of this cDNA coding are seen the sequence 2 in the sequence table, and the supposition molecular weight is 61.2KD.

Embodiment 3, Southern blot

Use DraI respectively, XspI, HaeIII enzyme cut 40 μ g New Zealand spinach genomic dnas, and these three kinds of enzymes do not have restriction enzyme site on clone's 535bpcDNA fragment.The DNA of complete degestion transfers to HybondN after 1.2% agarose electrophoresis is separated ⁺On the nylon membrane, press Random Primer DNA labeling Kit Ver.2 (TaKaRa, Japan) method with [ ³²P] the 725bp cDNA fragment of dCTP mark makees probe and carries out hybridization analysis.Found that DraI in the DNA that above-mentioned 3 kinds of enzymes are cut, XspI has 2 dna fragmentation hybridization, and as shown in Figure 2, HaeIII has 3 dna fragmentation hybridization, shows that the New Zealand spinach genome has the TtNHX1 of two to three

copies.Swimming lane

1,2,3 among Fig. 2 is represented respectively to use DraI, and XspI, HaeIII enzyme cut the Southern blot hybridization spot of New Zealand spinach genomic dna.

Embodiment 4, Northern blot

Carry out Northern blot experiment according to ordinary method, the result shows that the expression of TtNHX1 has tissue specificity as shown in Figure 3, at root, stem and the Ye Zhongjun of New Zealand spinach expression is arranged, and does not express in spending.Expression amount after NaCl induces in root, stem and the leaf increases, and does not still express in spending.Fig. 3 (a) is without inductive New Zealand spinach mRNA Northernblot hybridization spot, Fig. 3 (b) is the New Zealand spinach mRNA Northern blot hybridization spot after NaCl induces, and

swimming lane

1,2,3,4 is represented root, stem, leaf respectively and spent the middle mRNA Northern blot hybridization spot of extracting.

Embodiment 5, TtNHX1 sequential analysis

The hydrophobicity analysis that carries out with ordinary method shows that TtNHX1 has 12 and strides film district (as shown in Figure 4).The aminoacid sequence of TtNHX1 and other Na that has cloned ⁺/ H ⁺Reverse transport protein has very high homology, and TtNHX1 strides film district and other Na ⁺/ H ⁺Reverse transport protein to stride film district similarity higher.Among the TtNHX1 ⁸⁵LFFIYLLPPI ⁹⁴Be high conservative, this is Na ⁺/ H ⁺The binding site (as shown in Figure 5) that the competitive inhibitor ammonia chloropyrazine of reverse transport protein vigor is phonetic.Adding the blackboard branch among Fig. 5 is conforming amino acid, and what frame got up is the phonetic binding site of ammonia chloropyrazine.AgNHX1, AtNHX1, CNHX1, OsNHX1 and the TaNHX1 GenBank number of landing are respectively: BAB11940, AAF21755, AAK27314, BBA83337, AAK76737.

In order to analyze different N a ⁺/ H ⁺Genetic affinity between the reverse transport protein, the contriver has carried out Phylogenetic Analysis, with DNAMAN aminoacid sequence is analyzed.From GenBank, search some representational Na ⁺/ H ⁺The aminoacid sequence of reverse transport protein is analyzed TtNHX1 and their relation.The Na of TtNHX1 and vacuole type ⁺/ H ^-The relationship of reverse transport protein is near, and AgNHX1 is arranged, cNHX1, and AtNHX1, OsNHX1, TaNHX1, and NHXI, the function of AtNHX1 is with Na on vacuole skin ⁺Separating is in vacuole.TtNHX1 and AgNHX1 belong to same cluster, and TtNHX1 and AgNHX1 are the Na of halophytes ⁺/ H ⁺Reverse transport protein.The Na of TtNHX1 and plasma membrane type ⁺/ H ⁺The reverse transport protein sibship is (Fig. 6) far away.TtNHX1 is the Na of vacuole type as can be seen ⁺/ H ⁺Reverse transport protein.Above-mentioned reverse transport protein amino acid in the GenBank number of landing is: AgNHX1 (BAB11940), A.gmelini; OsNHX1 (BBA83337), O.sativa; TaNHX1 (AAK76737), T.aestivum; NHX1 (NP-010744), S.cerevisiae; Nhap (BAA316951), P.aeruginosa; SOS1 (AF256224), A.thaliana; NhaA (J03879), E.coli; Sod2 (CAA77796), S.pombe; NHA1 (NP-013239), S.cerevisiae.

The RT-PCR expression analysis of embodiment 6, TtNHX1

Expression with the RT-PCR methods analyst TtNHX1 of routine.The result as shown in Figure 7, TtNHX1 has expression in the New Zealand spinach adjoining tree, expression amount increases after NaCl induces, salt is handled the transcript of back TtNHX1 in New Zealand spinach and is increased sharply, and just can reach maintenance level in 1 hour.Illustrate that TtNHX1 may carry out the Na in the New Zealand spinach tenuigenin ⁺The function of ion transportation in the vacuole.

Sequence table

<160>2

<210>1

<211>2199

<212>DNA

＜213〉New Zealand spinach belongs to New Zealand spinach (Tetragonia tetragonioides O.)

<400>1

caaacataaa?aaaatagtcg?tatttcgggc?acgctgtgga?ttaagttatt?tgcatatata 60

gtggaagtac?aaggagttca?aaaacacaaa?aatggcattt?gatttgagct?attttgtgag 120

caacaagttg?ggaatgctgg?ccacttctga?tcatgcttct?gtggtatcta?tgaatctgtt 180

tgtggcgcta?ctgtgtggtt?gtatcgtgct?cggtcacctt?ctcgaggaga?atcgttggat 240

gaatgagtca?attacggctc?taattatagg?tttgggcact?ggagttgtga?ttttgctgat 300

tagcggcgga?aagagttcgc?atttgttggt?cttcagtgaa?gatcttttct?tcatatatct 360

tcttccgccg?attatattca?atgctgggtt?tcaggtgaag?aagaagcaat?ttttccgcaa 420

cttcatcaca?atcatactgt?ttggagctgt?gggtacattg?atatcattta?ccatcatatc 480

cctaggtgct?atggagttct?ttaagaagtt?ggacattggt?tctctggatt?taggcgacta 540

ccttgcaatt?ggtgcaatat?tcgctgcaac?cgattctgtt?tgcacgttgc?aggtgcttaa 600

tcaggacgag?acacctttgc?tatacagtct?ggtgtttggg?gaaggtgtag?ttaatgatgc 660

aacatcagtg?gtgcttttca?atgccatcca?gaactttgac?ctcacacata?ttgatcacag 720

aattgcttta?cagttcgctg?gcaatttcct?atatttattt?ttcacaagca?cgctgctagg 780

agcaatgact?ggattgctca?gtgcgtacat?tatcaaaaaa?ttgtactttg?gaaggcactc 840

cactgaccgt?gaagttgctc?taatgatgct?catggcgtat?ctatcgtaca?tgctcgctga 900

actattctat?ttgagtggaa?ttctgacagt?atttttctgt?gggattgtaa?tgtctcatta 960

tacatggcat?aacgtgactg?agagctcaag?agtaaccacc?aagcatgctt?tcgcaaccct 1020

gtcttttgtt?gccgagactt?tcatctttct?atatgtcggc?atggatgcat?tagacattga 1080

gaagtggaga?tttgtgagcg?atagtcctgg?aatatctgtt?gctgtgagtt?ccatattgct 1140

gggtctgctc?atgcttggac?gagcagcttt?tgtttctccc?ttgtccttcc?taatgaattt 1200

cttcaagaaa?tctcaagctg?aaaaagtcag?cttaaggcag?caggtgataa?tatggtgggc 1260

gggtctcatg?agaggtgctg?tttcgatggc?tcttgcttat?aatcagttta?cgaggtcagg 1320

gcacactcaa?ctaagaggga?atgcaataat?gattacgagc?actatatctg?ttgtccttgt 1380

cagcacagtg?gtatttggca?tactgacaaa?gcctcttata?ttgttcttgc?tgcctcaacc 1440

aaaacacttc?aatagtacca?gcactgtgtc?atcggaaaat?ttggggagcc?caaaatcatt 1500

cactttgcca?cttcttggta?atcaccaaga?ctctgaaacg?gatatcggaa?accatcaaga 1560

cagcactgac?aggggtcttg?gccggcccag?cagcctgcgc?atgcttctaa?atgcgccaag 1620

ccacacagtt?caccactact?ggcgcaaatt?cgataacgct?tttatgcggc?ctgtatttgg 1680

tggacgaggt?ttcgtacctt?atgtcccagg?ctcacctaca?gaacagagca?ccaacaattt 1740

gacagagagg?acataggaaa?gaaagagaga?cagttatggg?ataaatagaa?cctacaggca 1800

ggtttcaagg?tatagactgg?ccctcatcga?agaacatcga?ctggcttcat?cagctttgct 1860

gctgagagcc?tgagagcata?ttgtatatta?attttgttaa?tacataggtt?gtgcaactta 1920

cactgctcat?tgagttatgt?cattggactc?gaacaggatg?atcgtccaat?gtattctttg 1980

atttagctgt?agttcagctg?ccctaggatg?tttttggcgg?gttttagttg?ctttgtttta 2040

gacctagttt?cagggtttcc?ttctacaatc?tcttccccag?ctcggagttt?agtggtgtaa 2100

ttagtgtgga?ataatgtttg?ttcttgaaac?aagtccctgg?gaaacaattt?gggaaatagt 2160

tgcggtccaa?aaaaaaaaaa?aaaaaaaaaa?aaaaaaaaa 2199

<210>2

<211>554

<212>PRT

<400>2

Met?Ala?Phe?Asp?Leu?Ser?Tyr?Phe?Val?Ser?Asn?Lys?Leu?Gly?Met

1 5 10 15

Leu?Ala?Thr?Ser?Asp?His?Ala?Ser?Val?Val?Ser?Met?Asn?Leu?Phe

20 25 30

Val?Ala?Leu?Leu?Cys?Gly?Cys?Ile?Val?Leu?Gly?His?Leu?Leu?Glu

35 40 45

Glu?Asn?Arg?Trp?Met?Asn?Glu?Ser?Ile?Thr?Ala?Leu?Ile?Ile?Gly

50 55 60

Leu?Gly?Thr?Gly?Val?Val?Ile?Leu?Leu?Ile?Ser?Gly?Gly?Lys?Ser

65 70 75

Ser?His?Leu?Leu?Val?Phe?Ser?Glu?Asp?Leu?Phe?Phe?Ile?Tyr?Leu

80 85 90

Leu?Pro?Pro?Ile?Ile?Phe?Asn?Ala?Gly?Phe?Gln?Val?Lys?Lys?Lys

95 100 105

Gln?Phe?Phe?Arg?Asn?Phe?Ile?Thr?Ile?Ile?Leu?Phe?Gly?Ala?Val

110 115 120

Gly?Thr?Leu?Ile?Ser?Phe?Thr?Ile?Ile?Ser?Leu?Gly?Ala?Met?Glu

125 130 135

Phe?Phe?Lys?Lys?Leu?Asp?Ile?Gly?Ser?Leu?Asp?Leu?Gly?Asp?Tyr

140 145 150

Leu?Ala?Ile?Gly?Ala?Ile?Phe?Ala?Ala?Thr?Asp?Ser?Val?Cys?Thr

155 160 165

Leu?Gln?Val?Leu?Asn?Gln?Asp?Glu?Thr?Pro?Leu?Leu?Tyr?Ser?Leu

170 175 180

Val?Phe?Gly?Glu?Gly?Val?Val?Asn?Asp?Ala?Thr?Ser?Val?Val?Leu

185 190 195

Phe?Asn?Ala?Ile?Gln?Asn?Phe?Asp?Leu?Thr?His?Ile?Asp?His?Arg

200 205 210

Ile?Ala?Leu?Gln?Phe?Ala?Gly?Asn?Phe?Leu?Tyr?Leu?Phe?Phe?Thr

215 220 225

Ser?Thr?Leu?Leu?Gly?Ala?Met?Thr?Gly?Leu?Leu?Ser?Ala?Tyr?Ile

230 235 240

Ile?Lys?Lys?Leu?Tyr?Phe?Gly?Arg?His?Ser?Thr?Asp?Arg?Glu?Val

245 250 255

Ala?Leu?Met?Met?Leu?Met?Ala?Tyr?Leu?Ser?Tyr?Met?Leu?Ala?Glu

260 265 270

Leu?Phe?Tyr?Leu?Ser?Gly?Ile?Leu?Thr?Val?Phe?Phe?Cys?Gly?Ile

275 280 285

Val?Met?Ser?His?Tyr?Thr?Trp?His?Asn?Val?Thr?Glu?Ser?Ser?Arg

290 295 300

Val?Thr?Thr?Lys?His?Ala?Phe?Ala?Thr?Leu?Ser?Phe?Val?Ala?Glu

305 310 315

Thr?Phe?Ile?Phe?Leu?Tyr?Val?Gly?Met?Asp?Ala?Leu?Asp?Ile?Glu

320 325 330

Lys?Trp?Arg?Phe?Val?Ser?Asp?Ser?Pro?Gly?Ile?Ser?Val?Ala?Val

335 340 345

Ser?Ser?Ile?Leu?Leu?Gly?Leu?Leu?Met?Leu?Gly?Arg?Ala?Ala?Phe

350 355 360

Val?Ser?Pro?Leu?Ser?Phe?Leu?Met?Asn?Phe?Phe?Lys?Lys?Ser?Gln

365 370 375

Ala?Glu?Lys?Val?Ser?Leu?Arg?Gln?Gln?Val?Ile?Ile?Trp?Trp?Ala

380 385 390

Gly?Leu?Met?Arg?Gly?Ala?Val?Ser?Met?Ala?Leu?Ala?Tyr?Asn?Gln

395 400 405

Phe?Thr?Arg?Ser?Gly?His?Thr?Gln?Leu?Arg?Gly?Asn?Ala?Ile?Met

410 415 420

Ile?Thr?Ser?Thr?Ile?Ser?Val?Val?Leu?Val?Ser?Thr?Val?Val?Phe

425 430 435

Gly?Ile?Leu?Thr?Lys?Pro?Leu?Ile?Leu?Phe?Leu?Leu?Pro?Gln?Pro

440 445 450

Lys?His?Phe?Asn?Ser?Thr?Ser?Thr?Val?Ser?Ser?Glu?Asn?Leu?Gly

455 460 465

Ser?Pro?Lys?Ser?Phe?Thr?Leu?Pro?Leu?Leu?Gly?Asn?His?Gln?Asp

470 475 480

Ser?Glu?Thr?Asp?Ile?Gly?Asn?His?Gln?Asp?Ser?Thr?Asp?Arg?Gly

485 490 495

Leu?Gly?Arg?Pro?Ser?Ser?Leu?Arg?Met?Leu?Leu?Asn?Ala?Pro?Ser

500 505 510

His?Thr?Val?His?His?Tyr?Trp?Arg?Lys?Phe?Asp?Asn?Ala?Phe?Met

515 520 525

Arg?Pro?Val?Phe?Gly?Gly?Arg?Gly?Phe?Val?Pro?Tyr?Val?Pro?Gly

530 535 540

Ser?Pro?Thr?Glu?Gln?Ser?Thr?Asn?Asn?Leu?Thr?Glu?Arg?Thr

545 550 554

Claims

1, New Zealand spinach Na ⁺/ H ⁺Reverse transport protein TtNHX1, be protein, or the amino acid residue sequence of sequence 2 is passed through replacement, disappearance or the interpolation of one or several amino-acid residue and has identical active by sequence 2 deutero-protein with the amino acid residue sequence of sequence 2 with sequence 2 amino acid residue sequences in the sequence table.

2, New Zealand spinach Na according to claim 1 ⁺/ H ⁺Reverse transport protein is characterized in that: it is the protein with sequence 2 amino acid residue sequences in the sequence table.

3, New Zealand spinach Na ⁺/ H ⁺The encoding gene TtNHX1 of reverse transport protein is one of following nucleotide sequences:

1) the SEQ ID № in the sequence table: 1;

2) SEQ ID № in the code sequence tabulation: the polynucleotide of 2 protein sequences.

4, gene according to claim 3 is characterized in that: described New Zealand spinach Na ⁺/ H ⁺The encoding gene of reverse transport protein is the dna sequence dna of sequence 1 in the sequence table.

5, gene according to claim 4 is characterized in that: the reading frame of this gene is for holding the 92nd dna sequence dna to the 1756th bit base from 5 '.

6, contain the described expression carrier of claim 3.