CN1312174C - Sodium/hydrion inverse runner coding gene and application thereof - Google Patents
Sodium/hydrion inverse runner coding gene and application thereof Download PDFInfo
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- CN1312174C CN1312174C CNB021463107A CN02146310A CN1312174C CN 1312174 C CN1312174 C CN 1312174C CN B021463107 A CNB021463107 A CN B021463107A CN 02146310 A CN02146310 A CN 02146310A CN 1312174 C CN1312174 C CN 1312174C
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Abstract
本发明公开了一种钠/质子反向转运器及其编码基因与应用。本发明所提供的钠/质子反向转运器来源于玉米，名称为zmNHX1，是具有序列表中序列2氨基酸残基序列的蛋白质，或者是将序列2的氨基酸残基序列经过一个或几个氨基酸残基的取代、缺失或添加且具有与序列2的氨基酸残基序列相同活性的由序列2衍生的蛋白质。钠/质子反向转运器zmNHX1的编码基因，是下列核苷酸序列之一：1)序列表中序列1的DNA序列；2)与序列表中序列1限定的DNA序列具有90%以上同源性，且编码相同功能蛋白质的DNA序列。本发明的基因对培育抗盐植物品种，提高农作物产量具有重要意义。The invention discloses a sodium/proton antiporter, its coding gene and application. The sodium/proton antiporter provided by the present invention is derived from corn, and its name is zmNHX1. It is a protein with the amino acid residue sequence of sequence 2 in the sequence table, or the amino acid residue sequence of sequence 2 is passed through one or several amino acids A protein derived from SEQ ID NO: 2 that has the same activity as the amino acid residue sequence of SEQ ID NO: 2 by substitution, deletion or addition of residues. The coding gene of the sodium/proton antiporter zmNHX1 is one of the following nucleotide sequences: 1) the DNA sequence of sequence 1 in the sequence listing; 2) having more than 90% homology with the DNA sequence defined in sequence 1 in the sequence listing Sex, and the DNA sequence that encodes the same functional protein. The gene of the invention is of great significance for cultivating salt-resistant plant varieties and improving crop yield.
Description
Technical field
The present invention relates to sodium/proton antiport device and encoding gene and application in the plant genetic engineering field.
Background technology
Salt marsh has extremely agriculture production and seriously influences.Salt stress is to suppress plant-growth, reduces one of essential environmental factors of crop yield.The saltings is nearly 100,000,000 mu in 1,500,000,000 mu of arable lands of China, about 1,000,000,000 mu of middle-and-low-yielding fields, and major part is owing to due to arid saline and alkaline, also have 300,000,000 mu of saline-alkali wastelands in addition.And, irrigating regional secondary salinization field also in the continuation increase.
Na
+/ H
+Antiport device (Na
+/ H
+Antiporter) distribution is all arranged on cytolemma and vacuole skin.Na
+/ H
+The salt resistance of antiport device and halophytes has much relations, and it has guaranteed Na
+Compartmentation in vacuole greatly reduces the osmotic potential of vacuole, has alleviated Na again
+The injury of pair cell matter.Na
+/ H
+The antiport device can also be with the Na that enters in the root
+Pump, keep the ionic environment of lower concentration in the body, this mechanism is ubiquity in many farm crop.Na in the higher plant
+Effusive main mechanism is the H on the plasma membrane
+/ ATPase is H
+Pump cell, the electrochemical potential of generation can activate the Na on the plasma membrane
+/ H
+The antiport device, H
+Enter cell, Na
+Contrary electrochemical potential flows out.From the Physiology and biochemistry angle, on many higher plants and algae, all proved Na
+/ H
+The activity of antiport device.
The Na of many plants
+/ H
+The activity of antiport device carrier is very low, so they can finally death of dehydration in hypersaline environment.In hypersaline environment, Na
+/ H
+Synthetic and the activity of antiport device all can be suppressed, but by improving the content of unsaturated fatty acids on the cytolemma, can eliminate the influence of this hindering factor or increase Na by transgenic method
+/ H
+The content of antiport device in plant reaches the purpose of anti-salt.
Apse changed AtNHX1 in the Arabidopis thaliana over to agrobacterium mediation method in 1999, obtained anti-salt plant.This result has brought fine prospect for the anti-salt kind of cultivation.
Summary of the invention
The purpose of this invention is to provide sodium/proton antiport device and encoding gene thereof.
Sodium provided by the present invention/proton antiport device derives from corn, name is called zmNHX1, 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 539 amino-acid residues in the sequence table.
With corn EST design primer, be cloned into the full-length cDNA of zmNHX1 gene by 5 ', 3 ' RACE method.The encoding gene of sodium/proton antiport device zmNHX1 is one of following nucleotide sequences:
1) dna sequence dna of sequence 1 in the sequence table;
2) with sequence table in the dna sequence dna that limits of sequence 1 have 90% above homology, and the identical function protein DNA sequence of encoding.
The dna sequence dna of sequence 1 is by 2035 based compositions in the sequence table, and the reading frame of this gene is from 5 ' end the 1st to the 1617th bit base.
Sodium provided by the present invention/proton antiport device and encoding gene thereof can be from all unifacial leaves and dicotyledons, as tobacco, and Arabidopis thaliana, turfgrass, willow, soybean, wheat, paddy rice, cotton and tomato etc.Utilize any carrier that can guide foreign gene in plant, to express, gene transfered plant cell with coding sodium provided by the present invention/proton antiport device zmNHX1 can obtain high-salt stress tolerance enhanced transgenic cell line and transfer-gen plant.Gene of the present invention is in being building up to plant expression vector the time, before its transcription initiation Nucleotide, can add any enhancing promotor or inducible promoter, as cauliflower mosaic virus (CAMV) 35S promoter, the Ubiqutin promotor, enhanser both can be a transcriptional enhancer, also can be translational enhancer.For the ease of transgenic plant cells or plant being identified and screening, can process employed carrier, as the antibiotic marker thing (gentamicin, kantlex etc.) that adds the alternative mark (gus gene, luciferase genes etc.) of plant or have resistance.Carrying sodium of the present invention/proton antiport device zmNHX1 expression carrier can be Ti (the Tumor-induced-cancer is induced) plasmid, Ri (Root-induced-root induction) plasmid, plant viral vector etc., conversion can be passed through conventional biotechnological means such as agrobacterium-mediated transformation, particle bombardment, pollen tube passage method and realize, by the plant transformed host both can be monocotyledons, it also can be dicotyledons, as: paddy rice, wheat, corn, soybean, cotton, vegetables, trees and turfgrass etc.Gene pairs of the present invention is cultivated the salt-resistant plant kind, and it is significant to improve crop yield.
Description of drawings
Fig. 1 is Northern hybridization analysis result.
Embodiment
The clone of embodiment 1, sodium/proton antiport device zmNHX1
The corn of vegetative growth phase (growing for 2 weeks for 4 to 5 leaf phases) was handled 3 hours with 150mmNaCl.Extract the total RNA of root with TRIZLO (PROMAGA).Design and synthesize gene specific primer according to the corn est sequence
GSP1：5’agcgtgcacacagaatccgttgcag
GSP2：5’tgctgaatatcgctattgcgccgag
GSP3：5’ctcggcgcaatagcgatattcagca
GSP4：5’ctgcaacggattctgtgtgcacgct
Concrete operations are undertaken by SMART RACE (CLONTECH) test kit.At first carry out reverse transcription.Antimer is: 1ul salt is handled the total RNA of Zea mays root, 1ul 5 '-CDS or 3 '-CDS, 1ul SMATII OLIGO primer (10um/L), 2ul aqua sterilisa.Behind the mixing, in 70 ℃ of heating 2min.Put cooled on ice 2min, the centrifugal liquid that makes sinks to the bottom.Add the 2ul 5X first chain damping fluid, 1ul DTT (20mm/L), 1ul dNTP (10mm/L) and 1ul MMLV reversed transcriptive enzyme (20U/ul), 42 ℃ of reverse transcriptions 1.5 hours add the 100ul aqua sterilisa, heat the 2min termination reactions in 72 ℃.
Subsequently, with universal primer mixture (UPM) and GSP1, UPM and GSP3 carry out 5 ', 3 ' " TOUCHDOWN " pcr amplification respectively.Start the PCR reaction at PERKIN-ELMOR (PE) DNA THERMAL CYCLER 9700.Condition is 5X (94 ℃ of 30S, 72 ℃ of 3min), 5X (94 ℃ of 30S, 70 ℃ of 10S, 72 ℃ of 2min), 72 ℃ of min of 20X (94 ℃ of 30S, 63 ℃ of 30S, 72 ℃ of 30S).With this secondary response is template, uses universal primer (NUP) and GSP2 respectively, and GSP4 carries out nest-type PRC.5 ' RACE obtains the band of 400bp size, and 3 ' RACE obtains the band of 1.6Kb size.After reclaiming purifying respectively, 5 ' RACE fragment is connected on the PGEM-T EASY carrier, and 3 ' RACE directly checks order.Sequencing result and known est sequence splice, and proofread and correct in conjunction with order-checking peak figure.Obtain the good sequence zmNHX1 gene of splicing, shown in the sequence in the sequence table 1, total length is 2035bp altogether, and wherein 3 ' non-coding region is 418bp, and the coding region is 1617bp.Coded product is 539 amino acid, shown in the sequence in the sequence table 2.
Embodiment 2, the expression characteristic of zmNHX1 gene under the environment stress condition
The corn kind is in the flowerpot that contains vermiculite, and treat the long salt stress processing of wholeheartedly carrying out different concns to 3 leaves period: 50mM, 150mM NaCl waters to soil saturation.Collect after 3 hours, extract root, the total RNA of Ye, carry out Northern blot and analyze.The result as shown in Figure 3, R among the figure: the contrast root; L: contrast leaf; 50mmR:50mMNaCl handles root; 50mmL:50mM NaCl handles leaf; 150mmR:150mM NaCl handles root; 150mmL:150mMNaCl handles leaf.As can be seen from the figure, the zmNHX1 gene is expressed in root, does not express in leaf.After 50mMNaCl handled, the zmNHX1 expression amount raise significantly.When 100mM NaCl handles, the transcriptional level when the zmNHX1 transcriptional level is higher than 50mM NaCl processing.Show that zmNHX1 expresses and be subjected to Salt Stress-induced in root.
Sequence table
<160>2
<210>1
<211>2035
<212>DNA
＜213〉Zea corn (Zea mays.L)
<400>1
atggggctgg?gtgcgggggc?ggagctggtg?cggctgggcg?tgctgagctc?gacctctgac 60
cacgcgtcgg?tggtgtccat?caacctgttc?gtcgcgctgc?tctgcgcctg?catcgtcctg 120
ggccacctcc?tggaggagaa?ccgctgggtt?aacgagtcca?tcaccgcgct?catcatcggg 180
ctgtgcaccg?gcgtcgtgat?cctgctgacc?accaagggga?agagctcgca?catcctcgtc 240
ttcagcgagg?acctcttctt?catctacctc?ctacctccca?tcatcttcaa?tgccgagttc 300
caggtgaaga?agaaacggtt?cttccggaat?ttcatgacaa?tcacactgtt?gcagtgctgt 360
tggcacaatg?atatccttct?tcacatctct?ctcggcgcaa?tagcgatatt?cagcagaatg 420
aacattggga?cgctagatgt?cggggatttt?ctcgctattg?gagctatctt?ttctgcaacg 480
gattctgtgt?gcacgctgca?ggttctccat?caggatgaga?cgcccttttt?gtacagtctc 540
gtgttcggtg?agggagtcgt?gaacgatgcc?acgtctgttg?tgctcttcaa?cgcactccag 600
aacttcgatc?ttaaccacat?cgatgtagcc?gttgtgctga?atttcttggg?aaacttctgt 660
tacttattcg?tgtcaagcac?cttacttgga?gtgtttactg?gattgctcag?tgcctacata 720
attaagaagc?tatatatagg?aaggcattcc?actgaccgtg?aggttgcgct?tatgatgctc 780
atggcttacc?tctcctacat?gctggccgag?ctgctagatc?tgagtggcat?tcttaccgta 840
ttcttctgcg?gcatcgtgat?gtcgcattac?acctggcaca?atgtgacaga?gagctcaaga 900
gtcacaacca?agcatgcctt?tgctactttg?tccttcatcg?ccgagacttt?tctcttcctg 960
tatgttggga?tggatgccct?cgacatcgag?aagtgggagt?ttgccagtga?cagcccgggc 1020
aaatccattg?gcattagctc?gattttgcta?ggattggttc?tgctggggag?agctgcattt 1080
gttttcccat?tgtcgttttt?gtccaacctg?acaaagaagt?ctccattgga?gaaaataaca 1140
tttagacagc?aaattgtaat?atggtgggcc?ggactgatga?gaggtgccgt?gtccattgct 1200
ctcgcttaca?acaagttcac?gagatctgga?cacactgagc?tgcacggcaa?cgcgataatg 1260
atcaccagca?cgatcactgt?ggtcctgttt?agcactatgg?tgtttgggat?gatgacgaag 1320
ccattgatcc?ggctgctgct?ccctgcctgc?agcaacacgg?ccacctccga?gccgtcctca 1380
cccaagtccc?tgcactcgcc?tctcctgacg?agcatgcagg?gctcggacat?cgagacgggg 1440
tcggcacaga?ttgtgaggcc?gtccagcctc?cggatgctcc?taagcaagcc?aacccacacg 1500
gtgcactact?actggcgcaa?gttcgacgac?gcgctcatgc?ggcccatgtt?tggcggccgc 1560
gggttcgtgc?ccttctcccc?tggatcgccc?actgagcaga?gcgtccacga?cggacggtga 1620
acaaagccgc?gtctgacacc?aagagacgag?caggacatgc?ttttggctgc?taatggtcag 1680
atgatgatgt?ttttgtttgt?tgacagcttc?tgatggtgcg?ttagcaatgg?caaggaggtg 1740
tcagtttgtt?tggattagca?ggatgaagaa?gagcaaccct?ggtaactggg?gctgggggat 1800
tgagtttgtg?catgacaagg?ctgcaaactt?ttgtatgtag?ataaaaagaa?cacccatttg 1860
gccatttgta?ttgtatgtta?ttgtatgtaa?gctagcaggc?ctgtacaact?gcttttaggt 1920
cttcgtttgg?atgggcgaaa?ttctggcaaa?cagcagcaca?tccactctgt?gttctgatct 1980
aagcatcttg?gtttgatttt?tgaaatggac?tcggtgtgta?ggataagctt?ttgga 2035
<210>2
<211>539
<212>PRT
＜213〉Zea corn (Zea mays.L)
<400>2
Met?Gly?Leu?Gly?Ala?Gly?Ala?Glu?Leu?Val?Arg?Leu?Gly?Val?Leu
1 5 10 15
Ser?Ser?Thr?Ser?Asp?His?Ala?Ser?Val?Val?Ser?Ile?Asn?Leu?Phe
20 25 30
Val?Ala?Leu?Leu?Cys?Ala?Cys?Ile?Val?Leu?Gly?His?Leu?Leu?Glu
35 40 45
Glu?Asn?Arg?Trp?Val?Asn?Glu?Ser?Ile?Thr?Ala?Leu?Ile?Ile?Gly
50 55 60
Leu?Cys?Thr?Gly?Val?Val?Ile?Leu?Leu?Thr?Thr?Lys?Gly?Lys?Ser
65 70 75
Ser?His?Ile?Leu?Val?Phe?Ser?Glu?Asp?Leu?Phe?Phe?Ile?Tyr?Leu
80 85 90
Leu?Pro?Pro?Ile?Ile?Phe?Asn?Ala?Glu?Phe?Gln?Val?Lys?Lys?Lys
95 100 105
Arg?Phe?Phe?Arg?Asn?Phe?Met?Thr?Ile?Thr?Leu?Leu?Gln?Cys?Cys
110 115 120
Trp?His?Asn?Asp?Ile?Leu?Leu?His?Ile?Ser?Leu?Gly?Ala?Ile?Ala
125 130 135
Ile?Phe?Ser?Arg?Met?Asn?Ile?Gly?Thr?Leu?Asp?Val?Gly?Asp?Phe
140 145 150
Leu?Ala?Ile?Gly?Ala?Ile?Phe?Ser?Ala?Thr?Asp?Ser?Val?Cys?Thr
155 160 165
Leu?Gln?Val?Leu?His?Gln?Asp?Glu?Thr?Pro?Phe?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?Leu?Gln?Asn?Phe?Asp?Leu?Asn?His?Ile?Asp?Val?Ala
200 205 210
Val?Val?Leu?Asn?Phe?Leu?Gly?Asn?Phe?Cys?Tyr?Leu?Phe?Val?Ser
215 220 225
Ser?Thr?Leu?Leu?Gly?Val?Phe?Thr?Gly?Leu?Leu?Ser?Ala?Tyr?Ile
230 235 240
Ile?Lys?Lys?Leu?Tyr?Ile?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?Leu?Asp?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?Ile?Ala?Glu
305 310 315
Thr?Phe?Leu?Phe?Leu?Tyr?Val?Gly?Met?Asp?Ala?Leu?Asp?Ile?Glu
320 325 330
Lys?Trp?Glu?Phe?Ala?Ser?Asp?Ser?Pro?Gly?Lys?Ser?Ile?Gly?Ile
335 340 345
Ser?Ser?Ile?Leu?Leu?Gly?Leu?Val?Leu?Leu?Gly?Arg?Ala?Ala?Phe
350 355 360
Val?Phe?Pro?Leu?Ser?Phe?Leu?Ser?Asn?Leu?Thr?Lys?Lys?Ser?Pro
365 370 375
Leu?Glu?Lys?Ile?Thr?Phe?Arg?Gln?Gln?Ile?Val?Ile?Trp?Trp?Ala
380 385 390
Gly?Leu?Met?Arg?Gly?Ala?Val?Ser?Ile?Ala?Leu?Ala?Tyr?Asn?Lys
395 400 405
Phe?Thr?Arg?Ser?Gly?His?Thr?Glu?Leu?His?Gly?Asn?Ala?Ile?Met
410 415 420
Ile?Thr?Ser?Thr?Ile?Thr?Val?Val?Leu?Phe?Ser?Thr?Met?Val?Phe
425 430 435
Gly?Met?Met?Thr?Lys?Pro?Leu?Ile?Arg?Leu?Leu?Leu?Pro?Ala?Cys
440 445 450
Ser?Asn?Thr?Ala?Thr?Ser?Glu?Pro?Ser?Ser?Pro?Lys?Ser?Leu?His
455 460 465
Ser?Pro?Leu?Leu?Thr?Ser?Met?Gln?Gly?Ser?Asp?Ile?Glu?Thr?Gly
470 475 480
Ser?Ala?Gln?Ile?Val?Arg?Pro?Ser?Ser?Leu?Arg?Met?Leu?Leu?Ser
485 490 495
Lys?Pro?Thr?His?Thr?Val?His?Tyr?Tyr?Trp?Arg?Lys?Phe?Asp?Asp
500 505 510
Ala?Leu?Met?Arg?Pro?Met?Phe?Gly?Gly?Arg?Gly?Phe?Val?Pro?Phe
515 520 525
Ser?Pro?Gly?Ser?Pro?Thr?Glu?Gln?Ser?Val?His?Asp?Gly?Arg
530 535 539
Claims (7)
1, sodium/proton antiport device is the protein with sequence 2 amino acid residue sequences in the sequence table.
2, the encoding gene of the described sodium of claim 1/proton antiport device is one of following nucleotide sequences:
1) dna sequence dna of sequence 1 in the sequence table;
2) with sequence table in the dna sequence dna that limits of sequence 1 have 90% above homology, and the identical function protein DNA sequence of encoding.
3, gene according to claim 2 is characterized in that: the encoding gene of described sodium/proton antiport device is the dna sequence dna of sequence 1 in the sequence table.
4, gene according to claim 3 is characterized in that: the reading frame of this gene is for holding the 1st to the 1617th bit base from 5 '.
5, contain the described expression carrier of claim 3.
6, the transgenic cell line that contains the described gene of claim 3.
7, the application of the described gene of claim 3 in cultivating the salt-resistant plant kind.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB021463107A CN1312174C (en) | 2002-10-18 | 2002-10-18 | Sodium/hydrion inverse runner coding gene and application thereof |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB021463107A CN1312174C (en) | 2002-10-18 | 2002-10-18 | Sodium/hydrion inverse runner coding gene and application thereof |
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|---|---|
| CN1490331A CN1490331A (en) | 2004-04-21 |
| CN1312174C true CN1312174C (en) | 2007-04-25 |
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|---|---|---|---|
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| Country | Link |
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1204364A (en) * | 1995-10-12 | 1999-01-06 | 康乃尔研究基金会有限公司 | Production of Transgenic Cereal Plants Resistant to Drought or Salt Stress |
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2002
- 2002-10-18 CN CNB021463107A patent/CN1312174C/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1204364A (en) * | 1995-10-12 | 1999-01-06 | 康乃尔研究基金会有限公司 | Production of Transgenic Cereal Plants Resistant to Drought or Salt Stress |
Non-Patent Citations (1)
| Title |
|---|
| 植物盐胁迫应答的分子机制 沈义国，陈受宜，遗传，第23卷第4期 2001 * |
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|---|---|
| CN1490331A (en) | 2004-04-21 |
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