CN1425675A - Cotton Na+/H+ reverse transport protein gene and its cloning method and use - Google Patents
Cotton Na+/H+ reverse transport protein gene and its cloning method and use Download PDFInfo
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- CN1425675A CN1425675A CN 02151530 CN02151530A CN1425675A CN 1425675 A CN1425675 A CN 1425675A CN 02151530 CN02151530 CN 02151530 CN 02151530 A CN02151530 A CN 02151530A CN 1425675 A CN1425675 A CN 1425675A
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Abstract
The present invention relates to the cloning, recombination and salt tolerance analysis of cotton Na+/H+ reverse transport protein GhNHX1 gene, and belongs to the field of molecular biology and biological technology. Total RNA is extracted from cotton leaf treated with 0.4 mol/L NaCl solution and inversely transcribed into cDNA. Facultative primer is used for conventional PCR to obtain intermediate segment, and whole length cDNA is obtained through fast amplification in 3' and 5' ends. The gene is transformed to salt-sensitive yeast mutant to restore its salt tolerant capacity to some degree; and the gene is further transformed to tobacco to obtain transgenic plant capabl eof growing normally in salt concentration of 200 mmol/L. Therefore, the gene is one important salt tolerant gene and may be used in raising the salt tolerant capacity of plant for plantation in saline land.
Description
(1) technical field
The present invention relates to cotton Na
+/ H
+The analysis and the application of the clone of antiport protein gene GhNHX1, reorganization and salt tolerance function belong to molecular biology and biological technical field.
(2) background technology
The salt of high density causes that intravital ion imbalance of plant and height ooze coerces, and causes development of plants bad, and poor growth particularly reduces crop yield.Serious salt stress or osmotic stress can cause that plant intravital second coerces---the generation of oxidative stress, even cause plant death.Therefore, salt stress gets more and more people's extensive concerning, and the salt tolerance that improves crop also more and more is subject to people's attention.Under high salt, plant alleviates the murder by poisoning that salt stress causes by producing stress protein and solubility osmoregulation material.Early stage resistant gene of salt engineering mainly concentrates on to be removed free radical and increases by two aspects of osmoregulation material, and the salt resistance ability of transfer-gen plant increases, but effect is not too obvious.Recently, be positioned at Na on plasma membrane and the vacuole skin
+/ H
+The antiport protein gene is separated from many plants.Result of study shows the Na on the plasma membrane
+/ H
+Antiport albumen is responsible for Na
+Efflux, thereby keep low Na in the vegetable cell
+Level.And the Na on the vacuole skin
+/ H
+Antiport albumen then is responsible for Na
+The vacuole compartmentation, keep intracellular ion equilibrium.This has promoted the research work of resistant gene of salt engineering aspect greatly and has made a breakthrough.Blumwald etc. utilize genetic engineering means overexpression Na in tomato and rape
+The antiport protein gene, obtained first salt-tolerant plants truly in the world, be illustrated in accumulation salt in the anti-salt tomato plant of the transgenosis leaf of people such as Zhang Hongxia in calendar year 2001 " Nature Biotechnol) " and do not accumulate salt in the fruit, 19:765-768 (Hong-Xia Zhang, Eduardo Bhmmwald, 2001, Naturebiotechnology.19:765-768).
Because Na
+/ H
+The antiport protein gene is role in the salt tolerant process, and the inventor separates coding vacuole type Na from cotton
+/ H
+The proteic gene of antiport.According to the Na that delivers in other plant
+/ H
+The proteic aminoacid sequence of antiport, the design primer utilizes reverse transcription-polymerase chain reaction, isolates coding vacuole type Na from cotton
+/ H
+The proteic gene of antiport.Change this gene over to the salt sensitive yeast mutant, can recover its saline-alkaline tolerance to a certain extent.Further make up sense expression vector, transformation of tobacco, transfer-gen plant have higher salt tolerance, and salt tolerance can reach 200mmolL
-1This gene can be used for the genetic transformation of plant, improves the salt resistance ability of plant.
(3) summary of the invention
The present invention isolates coding vacuole type Na first from cotton
+/ H
+The proteic full-length cDNA of antiport is connected on the expression vector, utilizes Agrobacterium infestation method transformation of tobacco, and the transfer-gen plant salt tolerance of acquisition is up to 200mmolL
-1
From cotton leaf, extract total RNA, then the total RNA reverse transcription of 2 micrograms is become cDNA.According to the Na in other plant
+/ H
+The aminoacid sequence of guarding in the antiport albumen, design a pair of degenerate primer:
Forward primer: 5 '-CC (GATC) CC (GATC) AT (TAC) AT (TAC) TT (TC) AA (TC) GC-3 '
Reverse primer: 5 '-GT (GATC) AC (GATC) TT (AG) TGCCA (GATC) GT (AG) TA-3 '
Carry out conventional polymerase chain reaction (PCR), get 2 μ l PCR products and be connected on the pGEM-T easy carrier, transform DH5 α competent cell, be coated with flat board, screening positive clone is chosen hickie and is shaken bacterium, extract plasmid, and carry out enzyme and cut evaluation, carry out sequencing afterwards.Carry out 3 then ' and 5 ' terminal rapid amplifying obtain full-length cDNA.Concrete PCR reagent and condition are:
10 * reaction buffer, 5 μ l
25mM?MgCl
2 4μl
10mM deoxynucleoside acid mixture (dNTP) 1 μ l
Forward primer (10 μ M) 2 μ l
Reverse primer (10 μ M) 2 μ l
Taq archaeal dna polymerase 0.5 μ l
Cumulative volume 50 μ l
The PCR reaction conditions is: 94 ℃ 5 minutes, enter following circulation then: 94 ℃ 1 minute, 55 ℃ 1 minute, 72 ℃ 1 minute, totally 30 circulations, it is last 72 that (C extended 5 minutes.
The increase dna fragmentation of a 614bp of result.Get 2 μ l PCR products and be connected to pGEM-T easy carrier (Promega company product), transform DH5 α cell (common carrier host cell), dull and stereotyped incubated overnight.Picking white bacterial plaque is extracted plasmid DNA, is used for sequencing.
Through 3 ' and the cDNA that 5 ' terminal rapid amplifying obtains total length be 2485bp, comprise upstream sequence and poly A tract crust before the initiator codon.Open reading frame partly is 1629bp, push away thus 543 amino acid whose one section sequences of tool, this aminoacid sequence is compared in international gene pool, the proteic amino acid identity of Na+/H+ antiport of wild saltbush, paddy rice and the Arabidopis thaliana that shows and delivered is respectively 77.30%, 72.74% and 76.61%, shows through above-mentioned clone's step to have obtained the proteic gene of coding Na+/H+ antiport.This gene order is as follows: information (a) sequence signature of sequence table (1) SEQ ID NO 1
*Length: 2485 base pairs
*Type: nucleic acid
*Chain: two strands
*Topological framework: linear (b) molecule type: cDNA (c) supposes: deny (d) antisense: (e) is not initial originates: cotton (f) sequence description: SEQ IN N0.1
ACGCGGGGCAACACAGTCTTGATTTTGATCGTTTTTCGCTCCCATCGAAAGCGAAGATTT 60
TAAGCTGAAAAAAGAAGAGAGGAAAATTGTGGCAATTTGTTGGTGAGAAAGTCGAAGATT 120
CACGTGGGTAAGCTCCATAAACAGTGAAACATTGGATTTTCTTTTTTGTTTTTGTTTTCT 180
CAAGCTCTCTCTTCGAATTTACTCGTCTCTTTGAAACTGTCCGTTTTTTTTTGGTTCAAT 240
AAAATCGCAAATTATTTGCTAATTTAGAGAAGAAAATTGAACGGAGCTGAAACAAGGATG 300
ATTTGTTGCTGCATGATGTTGATTCTCCAAAACGATTCGAGTGCTTAAGGATTTTAAGAT 360
TAGAAAGTTCTTGAAATGGACAGTTCAGAGGCATAAAAATTTTCGAAGATTTACATTGTT 420
GAAGGAGAGCTTAAATCTGAAGCCTTGGACTACAACTGTTTCAGTTAGAAGGAATTGGTG 480
TTTAATAAAATTTGATTTAAAAAGAGGTCAATATGGTGGCTCCGCAGTTAGCTGCTGTCT 540
TTACTAAGTTGCAGACACTATCTACTTCAGACCATGCGTCTGTGGTCTCCATGAACATAT 600
TTGTAGCGCTTCTTTGTGCTTGCATTGTGATTGGTCATCTTTTGGAGGAGAATAGATGGA 660
TGAACGAATCAATTACTGCCCTTATCATTGGTGTTTTTACTGGGGTCATTATTTTGTTGA 720
CAAGTGGGGGTAAAAGCTCTCATCTTTTAGTCTTCAGTGAAGATCTGTTCTTTATCTATC 780
TTCTGCCCCCTATTATATTCAATGCTGGGTTTCAGGTGAAAAAGAAGCAATTTTTCCGTA 840
ACTTTATCACCATCATGCTGTTTGGGGCTGTTGGTACACTAATATCTTGTACAATTATCT 900
CTTTAGGTGTAATTAACTTCTTCAAGGAAATGGACATTGGCTCCTTAGACATTGGAGATT 960
TTCTAGCAATTGGTGCAATATTTGCTGCGACAGATTCTGTTTGCACACTGCAGGTGCTTA 1020
ATCAGGATGAGACTCCATTACTCTACAGTTTGGTTTTCGGAGAGGGTGTTGTAAATGATG 1080
CAACATCTGTGGTGCTTTTCAATGCAATCCAGAGTTTTGACCTCGTTAATACCAGTCCTA 1140
GAATTCTTCTGGAGTTTATTGGCAGCTTTTTGTATTTATTTTTAGCAAGCACTATGCTGG 1200
GAGTGATTGTTGGGTTGGTTAGTGCTTACATCATCAAAAAGTTGTACTTTGGAAGGCACT 1260
CAACAGATCGTGAATTTGCTCTTATGATGCTTATGGCATACCTTTCGTATATCATGGCTG 1320
AACTGTTCTATTTGAGTGGCATTCTTACAGTATTCTTTTGTGGGATTGTGATGTCACATT 1380
ATACCTGGCACAATGTAACTGAGAGTTCAAGAGTAACTACAAAGCATGCCTTTGCTACCT 1440
TGTCATTTGTTGCTGAGACTTTTCTCTTTCTTTATGTCGGGATGGATGCTTTGGACATGG 1500
AGAAGTGGAGATTTGTCAGTGATAGCCCTGGAACGTCAGTTGCTGTTAGTGCTGTGCTGA 1560
TGGGTCTTGTTATGGTTGGAAGAGCGGCTTTTGTGTTTCCCCTGTCATTTTTATCCAACT 1620
TGGCAAAGAAATCAACTAGTGAGAAAATCAGCTTCAGGGAACAAATTATAATATGGTGGG 1680
CTGGGCTCATGAGAGGCGCTGTATCTATGGCACTTGCATATAATCAGTTTACAAGGGGGG 1740
GCCATACTCAGTTGCGAGGAAATGCAATTATGATTACAAGCACCATAACCATTGTTCTAT 1800
TCAGCACTGTGGTTTTTGGTTTAATGACTAAACCTCTAATAAGGTTCTTGCTGCCTCATC 1860
CCAAACCAACAGCCAGCATGCTCTCAGACCAATCCACTCCAAAATCAATGGAGGCACCAT 1920
TTCTCGGAAGCGGCCAGGACTCTTTTGATGATAGTTTAATTGGAGTTCATCGACCAAACA 1880
GCATTCGTGCACTTCTTACAACTCCAGCACACACTGTTCATTACTATTGGCGAAAGTTTG 2040
ATAATGCCTTCATGCGCCCTATGTTTGGTGGCCGGGGTTTTGTGCCCTTCGTTCCTGGCT 2100
CCCCAACAGAAAGGAGTGAACCTAATCTGCCTCAATGGCAATGAGGTGGTTGAACAAGAT 2160
CTCTACAAAAATGTACATGTAATATAACAATGCAGTCGGTTGCAAAAAACATGCTTCTGG 2220
CGAGAAGCCAGTGCGGTATGCTTTGTATGTTTCATGTATAGGCTATATTTTGTTGGTTTT 2280
CAAGTTTCCTCAAGAGGTTCTTGTTTATTCTCCCCGAAACTACCTTCGCACCTGATGCTA 2340
TCTTTCCATTTGACATTTACGAATATTTATGATCTGGGTGAAGCTTAGGGGTAGGTGTGC 2400
CATTCTATTTTGTACGTATACGAGTATTTATTTTGTGTTTATATCAGTGTGTTTAGTTTT 2460
TATTTTTATTAAAAAAAAAAAAAAA 2485
(2) information of SEQ IN NO.2
(a) sequence signature
*Length: 543 amino acid
*Type: amino acid
*Chain: strand
*Topological framework: linearity
(b) molecule type: protein
( c ) MVAPQLAAVFTKLQTLSTSDHASVVSMNIFVALLCACIVIGHLLEENRWMNESITALIIG 60VFTGVIILLTSGGKSSHLLVFSEDLFFIYLLPPIIFNAGFQVKKKQFFRNFITIMLFGAV 120GTLISCTIISLGVINFFKEMDIGSLDIGDFLAIGAIFAATDSVCTLQVLNQDETPLLYSL 180VFGEGVVNDATSVVLFNAIQSFDLVNTSPRILLEFIGSFLYLFLASTMLGVIVGLVSAYI 240IKKLYFGRHSTDREFALMMLMAYLSYIMAELFYLSGILTVFFCGIVMSHYTWHNVTESSR 300VTTKHAFATLSFVAETFLFLYVGMDALDMEKWRFVSDSPGTSVAVSAVLMGLVMVGRAAF 360VFPLSFLSNLAKKSTSEKISFREQIIIWWAGLMRGAVSMALAYNQFTRGGHTQLRGNAIM 420ITSTITIVLFSTVVFGLMTKPLIRFLLPHPKPTASMLSDQSTPKSMEAPFLGSGQDSFDD 480SLIGVHRPNSIRALLTTPAHTVHYYWRKFDNAFMRPMFGGRGFVPFVPGSPTERSEPNLP 540QWQ 543,：
Forward primer: 5 '-ATGGTGGCTCCGCAGTTAGCT-3 '
Reverse primer: 5 '-ACCTCATTGCCATTGAGGCAG-3 '
CDNA with total RNA reverse transcription of leaf is a template, carries out the sequence that pcr amplification goes out to contain the total length encoder block, is connected on the pMD18-T carrier evaluation of checking order earlier.Downcut this fragment with XbaI and SalI then, be inserted into the downstream of the 35S promoter of expression vector PBI121.Change the expression vector that builds over to Agrobacterium EHAl05.
Transformation of tobacco screens containing on the LB solid medium of kantlex, with the resistance seedling that filters out at 100mmolL
-1, 200mmolL
-1And 30mmolL
-1Cultivate on the substratum of sodium-chlor.The result shows, compares with wild-type, and transfer-gen plant still can normal growth, the salt resistance ability that tool is higher.
The strain of transformed yeast salt sensitizing mutation is selecting to screen transformant on the substratum, and the positive transformant that filters out is cultivated crying out on the selection substratum that contains 1600mM sodium-chlor.The result shows that strain has compensating action to GhNHX1 to yeast salt sensitizing mutation, and the salt resistance ability that changes the yeast salt sensitizing mutation strain of 6hNHX1 strengthens greatly.
According to above-mentioned technology, from cotton, isolate coding vacuole type Na
+/ H
+The proteic gene GhNHX1 of antiport, this gene overexpression can cause the higher salt tolerance of transgene tobacco tool.Allow this gene in cotton, express, will improve the salt tolerance of cotton; If be connected with specific promoter, its expression is carried out space-time and stress-inducing is regulated and control, will have more specific aim, have very important economic benefit and social benefit.
(4) description of drawings Fig. 1 is that wild-type and the measurement result histogram X-coordinate that changes the photosynthetic rate of GhNHX1 gene plant are represented the NaCl concentration gradient, and ordinate zou is represented the photosynthetic rate value; W represents wild-type, and T represents transfer-gen plant, and mM represents mmol/L
-1Fig. 2 is that the measurement result histogram X-coordinate of the chlorophyll content of wild-type and transfer-gen plant is represented the NaCl concentration gradient, and ordinate zou is represented chlorophyll content; W represents wild-type, and T represents transfer-gen plant, and mM represents mmol/L
-1Fig. 3 is that the compensating action figure X-coordinate of GhNHX1 gene pairs yeast salt sensitive mutant is represented the NaC1 concentration gradient, and ordinate zou is represented the absorbance value at 600nm place; Control represents wild-type yeast bacterial strain K601, and R100 represents the responsive yeast mutant of salt, and the yeast strain of pYES2 (empty carrier) is changeed in the R100/pYES2 representative, and the yeast strain of GhNHX1 gene is changeed in R100/pYES2 GhNHX1 representative, and mM represents mmol/L
-1
(5) concrete invention embodiment embodiment 1: cotton Na
+/ H
+The cloning process of antiport protein gene
1. the extraction of total RNA: adopt RNAeasy mini kit (promoga company product) to extract total RNA.
2.cDNA article one chain is synthetic: get the total RNA of 2 micrograms, add 5 * reaction buffer, 4 μ l, 10mmolL
-1Thymus nucleic acid (dNTP) 2 μ l, ribonuclease inhibitor (40-200u/ μ 1) 0.5 μ l, primer T26 (10pmol/ μ 1) 1 μ l, ThermoScript II (10u/ μ 1) 2 μ l, 42 ℃ were reacted 85 ℃ of 10 minutes termination reactions 60 minutes.
3.PCR reaction: polymerase chain reaction (PCR) reagent and condition are:
At first following reagent is mixed:
10 * reaction buffer, 5 μ l
10mM deoxynucleoside acid mixture (dNTP) 1 μ l
Forward primer (10 μ molL
-1) 2 μ l
Reverse primer (10 μ molL
-1) 2 μ l
TaqDNA polysaccharase 0.5 μ l
Cumulative volume 50 μ l
The PCR reaction conditions is: 94 ℃ 5 minutes, enter following circulation then: 94 ℃ 1 minute, 55 ℃ 1 minute, 72 ℃ 1.5 minutes, totally 30 circulations, last 72 ℃ were extended 5 minutes.
4. gene clone: get 2 μ l PCR and be connected with pGEM-T easy carrier, operation steps is undertaken by Promega company product pGEM-T easy Vector system specification sheets.Connect product transformed into escherichia coli DH5 α bacterial strain then, scribble 5-bromo-4-chloro-3-indoles-β-D-galactoside on the surface) and the LB flat board that contains penbritin (100 mcg/ml) of X-gal on grow overnight.The picking white colony, overnight incubation in the LB liquid nutrient medium.
5. the extraction of plasmid DNA: alkaline process extracts plasmid DNA.
6. sequencing: originally be operated in Shanghai Sangon Biological Engineering Technology And Service Co., Ltd and carry out.
7.3 ' with the separating of 5 ' sequence: the SMART RACE cDNA Amplification Kit specification sheets by Clontech company carries out.
8. homology retrieval: utilize BLAST software that the sequence in isolated sequence and the gene library is compared.Embodiment 2: cotton Na
+/ H
+Antiport protein gene GhNHXl, following sequence:
(1) information of SEQ ID NO 1
(a) sequence signature
*Length: 2485 base pairs
*Type: nucleic acid
*Chain: two strands
*Topological framework: linearity
(b) molecule type: cDNA
(c) suppose: not
(d) antisense: not
(e) initial source: cotton
(f) sequence description: SEQ IN NO.1
ACGCGGGGCAACACAGTCTTGATTTTGATCGTTTTTCGCTCCCATCGAAAGCGAAGATTT 60
TAAGCTGAAAAAAGAAGAGAGGAAAATTGTGGCAATTTGTTGGTGAGAAAGTCGAAGATT 120
CACGTGGGTAAGCTCCATAAACAGTGAAACATTGGATTTTCTTTTTTGTTTTTGTTTTCT 180
CAAGCTCTCTCTTCGAATTTACTCGTCTCTTTGAAACTGTCCGTTTTTTTTTGGTTCAAT 240
AAAATCGCAAATTATTTGCTAATTTAGAGAAGAAAATTGAACGGAGCTGAAACAAGGATG 300
ATTTGTTGCTGCATGATGTTGATTCTCCAAAACGATTCGAGTGCTTAAGGATTTTAAGAT 360
TAGAAAGTTCTTGAAATGGACAGTTCAGAGGCATAAAAATTTTCGAAGATTTACATTGTT 420
GAAGGAGAGCTTAAATCTGAAGCCTTGGACTACAACTGTTTCAGTTAGAAGGAATTGGTG 480
TTTAATAAAATTTGATTTAAAAAGAGGTCAATATGGTGGCTCCGCAGTTAGCTGCTGTCT 540
TTACTAAGTTGCAGACACTATCTACTTCAGACCATGCGTCTGTGGTCTCCATGAACATAT 600
TTGTAGCGCTTCTTTGTGCTTGCATTGTGATTGGTCATCTTTTGGAGGAGAATAGATGGA 660
TGAACGAATCAATTACTGCCCTTATCATTGGTGTTTTTACTGGGGTCATTATTTTGTTGA 720
CAAGTGGGGGTAAAAGCTCTCATCTTTTAGTCTTCAGTGAAGATCTGTTCTTTATCTATC 780
TTCTGCCCCCTATTATATTCAATGCTGGGTTTCAGGTGAAAAAGAAGCAATTTTTCCGTA 840
ACTTTATCACCATCATGCTGTTTGGGGCTGTTGGTACACTAATATCTTGTACAATTATCT 900
CTTTAGGTGTAATTAACTTCTTCAAGGAAATGGACATTGGCTCCTTAGACATTGGAGATT 960
TTCTAGCAATTGGTGCAATATTTGCTGCGACAGATTCTGTTTGCACACTGCAGGTGCTTA 1020
ATCAGGATGAGACTCCATTACTCTACAGTTTGGTTTTCGGAGAGGGTGTTGTAAATGATG 1080
CAACATCTGTGGTGCTTTTCAATGCAATCCAGAGTTTTGACCTCGTTAATACCAGTCCTA 1140
GAATTCTTCTGGAGTTTATTGGCAGCTTTTTGTATTTATTTTTAGCAAGCACTATGCTGG 1200
GAGTGATTGTTGGGTTGGTTAGTGCTTACATCATCAAAAAGTTGTACTTTGGAAGGCACT 1260
CAACAGATCGTGAATTTGCTCTTATGATGCTTATGGCATACCTTTCGTATATCATGGCTG 1320
AACTGTTCTATTTGAGTGGCATTCTTACAGTATTCTTTTGTGGGATTGTGATGTCACATT 1380
ATACCTGGCACAATGTAACTGAGAGTTCAAGAGTAACTACAAAGCATGCCTTTGCTACCT 1440
TGTCATTTGTTGCTGAGACTTTTCTCTTTCTTTATGTCGGGATGGATGCTTTGGACATGG 1500
AGAAGTGGAGATTTGTCAGTGATAGCCCTGGAACGTCAGTTGCTGTTAGTGCTGTGCTGA 1560
TGGGTCTTGTTATGGTTGGAAGAGCGGCTTTTGTGTTTCCCCTGTCATTTTTATCCAACT 1620
TGGCAAAGAAATCAACTAGTGAGAAAATCAGCTTCAGGGAACAAATTATAATATGGTGGG 1680
CTGGGCTCATGAGAGGCGCTGTATCTATGGCACTTGCATATAATCAGTTTACAAGGGGGG 1740
GCCATACTCAGTTGCGAGGAAATGCAATTATGATTACAAGCACCATAACCATTGTTCTAT 1800
TCAGCACTGTGGTTTTTGGTTTAATGACTAAACCTCTAATAAGGTTCTTGCTGCCTCATC 1860
CCAAACCAACAGCCAGCATGCTCTCAGACCAATCCACTCCAAAATCAATGGAGGCACCAT 1920
TTCTCGGAAGCGGCCAGGACTCTTTTGATGATAGTTTAATTGGAGTTCATCGACCAAACA 1880
GCATTCGTGCACTTCTTACAACTCCAGCACACACTGTTCATTACTATTGGCGAAAGTTTG 2040
ATAATGCCTTCATGCGCCCTATGTTTGGTGGCCGGGGTTTTGTGCCCTTCGTTCCTGGCT 2100
CCCCAACAGAAAGGAGTGAACCTAATCTGCCTCAATGGCAATGAGGTGGTTGAACAAGAT 2160
CTCTACAAAAATGTACATGTAATATAACAATGCAGTCGGTTGCAAAAAACATGCTTCTGG 2220
CGAGAAGCCAGTGCGGTATGCTTTGTATGTTTCATGTATAGGCTATATTTTGTTGGTTTT 2280
CAAGTTTCCTCAAGAGGTTCTTGTTTATTCTCCCCGAAACTACCTTCGCACCTGATGCTA 2340
TCTTTCCATTTGACATTTACGAATATTTATGATCTGGGTGAAGCTTAGGGGTAGGTGTGC 2400
CATTCTATTTTGTACGTATACGAGTATTTATTTTGTGTTTATATCAGTGTGTTTAGTTTT 2460
Information (a) sequence signature of TATTTTTATTAAAAAAAAAAAAAAA 2485 (3) SEQ IN NO.2
*Length: 543 amino acid
*Type: amino acid
*Chain: strand
*Topological framework: linearity
(b) molecule type: protein
( c ) MVAPQLAAVFTKLQTLSTSDHASVVSMNIFVALLCACIVIGHLLEENRWMNESITALIIG 60VFTGVIILLTSGGKSSHLLVFSEDLFFIYLLPPIIFNAGFQVKKKQFFRNFITIMLFGAV 120GTLISCTIISLGVINFFKEMDIGSLDIGDFLAIGAIFAATDSVCTLQVLNQDETPLLYSL 180VFGEGVVNDATSVVLFNAIQSFDLVNTSPRILLEFIGSFLYLFLASTMLGVIVGLVSAYI 240IKKLYFGRHSTDREFALMMLMAYLSYIMAELFYLSGILTVFFCGIVMSHYTWHNVTESSR 300VTTKHAFATLSFVAETFLFLYVGMDALDMEKWRFVSDSPGTSVAVSAVLMGLVMVGRAAF 360VFPLSFLSNLAKKSTSEKISFREQIIIWWAGLMRGAVSMALAYNQFTRGGHTQLRGNAIM 420ITSTITIVLFSTVVFGLMTKPLIRFLLPHPKPTASMLSDQSTPKSMEAPFLGSGQDSFDD 480SLIGVHRPNSIRALLTTPAHTVHYYWRKFDNAFMRPMFGGRGFVPFVPGSPTERSEPNLP 540QWQ 5433：
1. according to isolated cotton Na
+/ H
+The nucleotide sequence of antiport protein gene GhNHXl, the design primer:
Forward primer: 5 '-ATGGTGGCTCCGCAGTTAGCT-3 '
Reverse primer: 5 '-ACCTCATTGCCATTGAGGCAG-3 '
CDNA with total RNA reverse transcription of leaf is a template, carries out the polymerase chain reaction.
2. get 2 μ l PCR and be connected with the pMD18-T carrier, operation steps is undertaken by Promega company product pMD18-T Vectorsystem specification sheets.Transformed into escherichia coli DH5 α bacterial strain then is coated with grow overnight on the LB flat board that contains penbritin (100 mcg/ml) of 5-bromo-4-chloro-3-indoles-β-D-galactoside and X-gal on the surface.The picking white colony, overnight incubation in the LB liquid nutrient medium.Alkaline process extracts plasmid DNA, carries out sequencing.
3. with X α bI and two restriction enzymes of S α lI this gene is downcut from the pMD18-T carrier, the PBI121 that cuts with the same enzyme enzyme is connected.Connect product and transform DH5 α cell, cultivate containing on the LB solid plate of penbritin then, bacterium colony is carried out PCR identifies and the restriction analysis of plasmid DNA.
4. the expression vector that builds is transformed Agrobacterium EHAl05.Embodiment 4: the gene salt tolerance is analyzed
1. plant and plant tobacco.
2. the Agrobacterium mono-clonal identified of picking is in the LB liquid nutrient medium that contains 50 mg/litre kantlex, 28 ℃ of shaking culture.
3.3000rpm centrifugal 5 minutes, the Agrobacterium precipitation suspended with 10X MS substratum.
4. tobacco leaf is cut into small pieces, puts into above-mentioned suspension and soaked 10 minutes.
5. the blade stripping and slicing after infecting places division culture medium (1 * MS salt, 3% sucrose, pH5.8,4.5g/L carrageenin,) on cultivated altogether 2 days, change over to then and select substratum (1 * MS salt, 3% sucrose, pH5.8,4.5g/L carrageenin, kantlex 100 mg/litre, cephalo penicillin 250 mg/litre) screening, obtain resistant plant.
6. resistant plant is moved into flowerpot, watered in per two days and to contain 10mmolL
-1, 100mmolL
-1, 200mmolL
-1And 300mmolL
-1The 1/2Hoagland nutritive medium of sodium-chlor, the photosynthetic rate and the chlorophyll content of mensuration wild-type and transfer-gen plant after 20 days.Embodiment 5: cotton Na
+/ H
+Antiport protein gene GhNHX1 is to the compensating action of yeast salt sensitizing mutation strain
With the full-length cDNA subclone of being cloned in the pMD18-T carrier, be built into pMD18-T-GhNHX1.
2. pMD18-T-GhNHX1 and Yeast expression carrier pYES2 are cut through BamHI and HindIII enzyme, reclaim the purpose band, under the ligase enzyme effect, be built into the pYES2-GhNHX1 expression vector.
3. in yeast strain K601 (wild-type) on the picking YPD flat board and R100 (strain of salt sensitizing mutation) single bacterium colony and the 5mlYPD nutrient solution, 30 ℃ of shaking culture are spent the night, 10 times of redilution, shaking culture 6 hours, 3000rpm collected thalline in centrifugal 5 minutes, with the resuspended thalline of 1.5mlTE.
4. get the above-mentioned bacterium liquid of 0.1ml, 3 μ g pYES2-GhNHX1 plasmids and 0.6mlTE-liOAC-PEG solution respectively in a 1.5ml centrifuge tube, 30 ℃ of shaking culture 30 minutes, 42 ℃ of thermal shocks 15 minutes, 3000rpm collected thalline in centrifugal 20 seconds, be resuspended in the 0.5ml redistilled water, get 200 μ l and coat on the selection substratum, cultivated 2-4 days for 30 ℃.
5. transformant is inoculated into and selects in the nutrient solution, and 30 ℃ of shaking culture 48 hours are measured the photoabsorption at 600nm place.
Sodium ion antiport protein gene sequence table-word
SEQUENCE LISTING＜110〉＜120〉Na+/H+＜130〉1＜160〉2＜170〉PatentIn version 3.1＜210〉1＜211〉2485＜212〉DNA＜213〉Gossypium hirsutum＜220〉1＜221〉CDS＜222〉 ( 513 ) .. ( 2141 )＜223〉＜220〉＜221〉5’UTR＜222〉 ( 1 ) .. ( 512 )＜223〉＜220〉＜221〉3’UTR＜222〉 ( 2142 ) .. ( 2485 )＜223〉＜400〉1acgcggggca acacagtctt gattttgatc gtttttcgct cccatcgaaa gcgaagattt 60taagctgaaa aaagaagaga ggaaaattgt ggcaatttgt tggtgagaaa gtcgaagatt 120cacgtgggta agctccataa acagtgaaac attggatttt cttttttgtt tttgttttct 180caagctctct cttcgaattt actcgtctct ttgaaactgt ccgttttttt ttggttcaat 240aaaatcgcaa attatttgct aatttagaga agaaaattga acggagctga aacaaggatg 300atttgttgct gcatgatgtt gattctccaa aacgattcga gtgcttaagg attttaagat 360tagaaagttc ttgaaatgga cagttcagag gcataaaaat tttcgaagat ttacattgtt 420gaaggagagc ttaaatctga agccttggac tacaactgtt tcagttagaa ggaattggtg 480tttaataaaa tttgatttaa aaagaggtca at atg gtg gct ccg cag tta gct 533
Met?Val?Ala?Pro?Gln?Leu?Ala
1 5gct?gtc?ttt?act?aag?ttg?cag?aca?cta?tct?act?tca?gac?cat?gcg?tct 581Ala?Val?Phe?Thr?Lys?Leu?Gln?Thr?Leu?Ser?Thr?Ser?Asp?His?Ala?Ser
10 15 20gtg?gtc?tcc?atg?aac?ata?ttt?gta?gcg?ctt?ctt?tgt?gct?tgc?att?gtg 629Val?Val?Ser?Met?Asn?Ile?Phe?Val?Ala?Leu?Leu?Cys?Ala?Cys?Ile?Val
25 30 35att?ggt?cat?ctt?ttg?gag?gag?aat?aga?tgg?atg?aac?gaa?rca?att?act 677Ile?Gly?His?Leu?Leu?Glu?Glu?Asn?Arg?Trp?Met?Ash?Glu?Ser?Ile?Thr40 45 50 55gcc?ctt?atc?att?ggt?gtt?ttt?act?ggg?gtc?att?att?ttg?ttg?aca?agt 725Ala?Leu?Ile?Ile?Gly?Val?Phe?Thr?Gly?Val?Ile?Ile?Leu?Leu?Thr?Ser
60 65 70ggg?ggt?aaa?agc?tct?cat?ctt?tta?gtc?ttc?agt?gaa?gat?ctg?ttc?ttt 773Gly?Gly?Lys?Ser?Ser?His?Leu?Leu?Val?Phe?Set?Glu?Asp?Leu?Phe?Phe
75 80 85atc?tat?ctt?ctg?ccc?cct?att?ata?ttc?aat?gct?ggg?ttt?cag?gtg?aaa 821Ile?Tyr?Leu?Leu?Pro?Pro?Ile?Ile?Phe?Ash?Ala?Gly?Phe?Gln?Val?Lys
90 95 100aag?aag?caa?ttt?ttc?cgt?aac?ttt?atc?acc?atc?atg?ctg?ttt?ggg?gct 869Lys?Lys?Gln?Phe?Phe?Arg?Asn?Phe?Ile?Thr?Ile?Met?Leu?Phe?Gly?Ala
105 110 115gtt?ggt?aca?cta?ata?tct?tgt?aca?att?atc?tct?tta?ggt?gta?att?aac 917Val?Gly?Thr?Leu?Ile?Ser?Cys?Thr?Ile?Ile?Ser?Leu?Gly?Val?Ile?Asn120 125 130 135ttc?ttc?aag?gaa?atg?gac?att?ggc?tcc?tta?gac?att?gga?gat?ttt?cta 965Phe?Phe?Lys?Glu?Met?Asp?Ile?Gly?Ser?Leu?Asp?lle?Gly?Asp?Phe?Leu
140 145 150gca?att?ggt?gca?ata?ttt?gct?gcg?aca?gat?tct?gtt?tgc?aca?ctg?cag 1013Ala?Ile?Gly?Ala?Ile?Phe?Ala?Ala?Thr?Asp?Ser?Val?Cys?Thr?Leu?Gln
155 160 165gtg?ctt?aat?cag?gat?gag?act?cca?tta?ctc?tac?agt?ttg?gtt?ttc?gga 1061Val?Leu?Asn?Gln?Asp?Glu?Thr?Pro?Leu?Leu?Tyr?Ser?Leu?Val?Phe?Gly
170 175 180gag?ggt?gtt?gta?aat?gat?gca?aca?tct?gtg?gtg?ctt?ttc?aat?gca?atc 1109Glu?Gly?Val?Val?Asn?Asp?Ala?Thr?Ser?Val?Val?Leu?Phe?Asn?Ala?Ile
185 190 195cag?agt?ttt?gac?ctc?gtt?aat?acc?agt?cct?aga?att?ctt?ctg?gag?ttt 1157Gln?Ser?Phe?Asp?Leu?Val?Asn?Thr?Ser?Pro?Arg?Ile?Leu?Leu?Glu?Phe200 205 210 215att?ggc?agc?ttt?ttg?tat?tta?ttt?tta?gca?agc?act?atg?ctg?gga?gtg 1205Ile?Gly?Ser?Phe?Leu?Tyr?Leu?Phe?Leu?Ala?Ser?Thr?Met?Leu?Gly?Val
220 225 230att?gtt?ggg?ttg?gtt?agt?gct?tac?atc?atc?aaa?aag?ttg?tac?ttt?gga 1253
Sodium ion antiport protein gene sequence table-wordIle Val Gly Leu Val Ser Ala Tyr Ile Ile Lys Lys Leu Tyr Phe Gly
235 240 245agg?cac?tca?aca?gat?cgt?gaa?ttt?gct?ctt?atg?atg?ctt?atg?gca?tac 1301Arg?His?Ser?Thr?Asp?Arg?Glu?Phe?Ala?Leu?Met?Met?Leu?Met?Ala?Tyr
250 255 260ctt?tcg?tat?atc?atg?gct?gaa?ctg?ttc?tat?ttg?agt?ggc?att?ctt?aca 1349Leu?Ser?Tyr?Ile?Met?Ala?Glu?Leu?Phe?Tyr?Leu?Ser?Gly?Ile?Leu?Thr
265 270 275gta?ttc?ttt?tgt?ggg?att?gtg?atg?tca?cat?tat?acc?tgg?cac?aat?gta 1397Val?Phe?Phe?Cys?Gly?Ile?Val?Met?Ser?His?Tyr?Thr?Trp?His?Ash?Val280 285 290 295act?gag?agt?tca?aga?gta?act?aca?aag?cat?gcc?ttt?gct?acc?ttg?tca 1445Thr?Glu?Ser?Ser?Arg?Val?Thr?Thr?Lys?His?Ala?Phe?Ala?Thr?Leu?Ser
300 305 310ttt?gtt?gct?gag?act?ttt?ctc?ttt?ctt?tat?gtc?ggg?atg?gat?gct?ttg 1493Phe?Val?Ala?Glu?Thr?Phe?Leu?Phe?Leu?Tyr?Val?Gly?Met?Asp?Ala?Leu
315 320 325gac?atg?gag?aag?tgg?aga?ttt?gtc?agt?gat?agc?cct?gga?acg?tca?gtt 1541Asp?Met?Glu?gys?Trp?Arg?Phe?Val?Ser?Asp?Ser?Pro?Gly?Thr?Ser?Val
330 335 340gct?gtt?agt?gct?gtg?ctg?atg?ggt?ctt?gtt?atg?gtt?gga?aga?gcg?gct 1589Ala?Val?Ser?Ala?Val?Leu?Met?Gly?Leu?Val?Met?Val?Gly?Arg?Ala?Ala
345 350 355ttt?gtg?ttt?ccc?ctg?tca?ttt?tta?tcc?aac?ttg?gca?aag?aaa?tca?act 1637Phe?Val?Phe?Pro?Leu?Ser?Phe?Leu?Ser?Asn?Leu?Ala?Lys?Lys?Ser?Thr360 365 370 375agt?gag?aaa?atc?agc?ttc?agg?gaa?caa?att?ata?ata?tgg?tgg?gct?ggg 1685Ser?Glu?Lys?Ile?Ser?Phe?Arg?Glu?Gln?Ile?Ile?Ile?Trp?Trp?Ala?Gly
380 385 390ctc?atg?aga?ggc?gct?gta?tct?atg?gca?ctt?gca?tat?aat?cag?ttt?aca 1733Leu?Met?Arg?Gly?Ala?Val?Ser?Met?Ala?Leu?Ala?Tyr?Ash?Gln?Phe?Thr
395 400 405agg?ggg?ggc?cat?act?cag?ttg?cga?gga?aat?gca?att?atg?att?aca?agc 1781Arg?Gly?Gly?His?Thr?Gln?Leu?Arg?Gly?Ash?Ala?Ile?Met?Ile?Thr?Ser
410 415 420acc?ata?acc?att?gtt?cta?ttc?agc?act?gtg?gtt?ttt?ggt?tta?atg?act 1829Thr?Ile?Thr?Ile?Val?Leu?Phe?Ser?Thr?Val?Val?Phe?Gly?Leu?Met?Thr
425 430 435aaa?cct?cta?ata?agg?ttc?ttg?ctg?cct?cat?ccc?aaa?cca?aca?gcc?agc 1877Lys?Pro?Leu?Ile?Arg?Phe?Leu?Leu?Pro?His?Pro?Lys?Pro?Thr?Ala?Set440 445 450 455atg?ctc?tca?gac?caa?tcc?act?cca?aaa?tca?atg?gag?gca?cca?ttt?ctc 1925Met?Leu?Ser?Asp?Gln?Ser?Thr?Pro?Lys?Ser?Met?Glu?Ala?Pro?Phe?Leu
460 465 470gga?agc?ggc?cag?gac?tct?ttt?gat?gat?agt?tta?att?gga?gtt?cat?cga 1973Gly?Ser?Gly?Gln?Asp?Ser?Phe?Asp?Asp?Ser?Leu?Ile?Gly?Val?His?Arg
475 480 485cca?aac?agc?att?cgt?gca?ctt?ctt?aca?act?cca?gca?cac?act?gtt?cat 2021Pro?Asn?Ser?Ile?Arg?Ala?Leu?Leu?Thr?Thr?Pro?Ala?His?Thr?Val?His
490 495 500tac?tat?tgg?cga?aag?ttt?gat?aat?gcc?ttc?atg?cgc?cct?atg?ttt?ggt 2069Tyr?Tyr?Trp?Arg?Lys?Phe?Asp?Ash?Ala?Phe?Met?Arg?Pro?Met?Phe?Gly
505 510 515ggc?cgg?ggt?ttt?gtg?ccc?ttc?gtt?cct?ggc?tcc?cca?aca?gaa?agg?agt 2117Gly?Arg?Gly?Phe?Val?Pro?Phe?Val?Pro?Gly?Ser?Pro?Thr?Glu?Arg?Ser520 525 530 535gaa?cct?aat?ctg?cct?caa?tgg?caa?tgaggtggtt?gaacaagatc?tctacaaaaa 2171Glu?Pro?Asn?Leu?Pro?Gln?Trp?Gln
540tgtacatgta?atataacaat?gcagtcggtt?gcaaaaaaca?tgcttctggc?gagaagccag 2231tgcggtatgc?tttgtatgtt?tcatgtatag?gctatatttt?gttggttttc?aagtttcctc 2291aagaggttct?tgtttattct?ccccgaaact?accttcgcac?ctgatgctat?ctttccattt 2351gacatttacg?aatatttatg?atctgggtga?agcttagggg?taggtgtgcc?attctatttt 2411gtacgtatac?gagtatttat?tttgtgttta?tatcagtgtg?tttagttttt?atttttatta 2471aaaaaaaaaa?aaaa 2485<210>2<211>543<212>PRT<213>Gossypium?hirsutum<400>2Met?Val?Ala?Pro?Gln?Leu?Ala?Ala?Val?Phe?Thr?Lys?Leu?Gln?Thr?Leu1 5 10 15Ser?Thr?Ser?Asp?His?Ala?Ser?Val?Val?Ser?Met?Asn?Ile?Phe?Val?Ala
20 25 30
Sodium ion antiport protein gene sequence table-wordLeu Leu Cys Ala Cys Ile Val Ile Gly His Leu Leu Glu Glu Asn Arg
35 40 45Trp?Met?Asn?Glu?Ser?Ile?Thr?Ala?Leu?Ile?Ile?Gly?Val?Phe?Thr?Gly
50 55 60Val?Ile?Ile?Leu?Leu?Thr?Ser?Gly?Gly?Lys?Ser?Ser?His?Leu?Leu?Val65 70 75 80Phe?Ser?Glu?Asp?Leu?Phe?Phe?Ile?Tyr?Leu?Leu?Pro?Pro?Ile?Ile?Phe
85 90 95Asn?Ala?Gly?Phe?Gln?ValLys?Lys?Lys?Gln?Phe?Phe?Arg?Asn?Phe?Ile
100 105 110Thr?Ile?Met?Leu?Phe?Gly?Ala?Val?Gly?Thr?Leu?Ile?Ser?Cys?Thr?Ile
115 120 125Ile?Ser?Leu?Gly?Val?Ile?Asn?Phe?Phe?Lys?Glu?Met?Asp?Ile?Gly?Ser
130 135 140Leu?Asp?Ile?Gly?Asp?Phe?Leu?Ala?Ile?Gly?Ala?Ile?Phe?Ala?Ala?Thr145 150 155 160Asp?Ser?Val?Cys?Thr?Leu?Gln?Val?Leu?Asn?Gln?Asp?Glu?Thr?Pro?Leu
165 170 175Leu?Tyr?Ser?Leu?Val?Phe?Gly?Glu?Gly?Val?Val?Asn?Asp?Ala?Thr?Ser
180 185 190Val?Val?Leu?Phe?Asn?Ala?Ile?Gln?Ser?Phe?Asp?Leu?Val?Asn?Thr?Ser
195 200 205Pro?Arg?Ile?Leu?Leu?Glu?Phe?Ile?Gly?Ser?Phe?Leu?Tyr?Leu?Phe?Leu
210 215 220Ala?Ser?Thr?Met?Leu?Gly?Val?Ile?Val?Gly?Leu?Val?Ser?Ala?Tyr?Ile225 230 235 240Ile?Lys?Lys?Leu?Tyr?Phe?Gly?Arg?His?Ser?Thr?Asp?Arg?Glu?Phe?Ala
245 250 255Leu?Met?Met?Leu?Met?Ala?Tyr?Leu?Ser?Tyr?Ile?Met?Ala?Glu?Leu?Phe
260 265 270Tyr?Leu?Ser?Gly?Ile?Leu?Thr?Val?Phe?Phe?Cys?Gly?Ile?Val?Met?Ser
275 280 285His?Tyr?Thr?Trp?His?Asn?Val?Thr?Glu?Ser?Ser?Arg?Val?Thr?Thr?Lys
290 295 300His?Ala?Phe?Ala?Thr?Leu?Ser?Phe?Val?Ala?Glu?Thr?Phe?Leu?Phe?Leu305 310 315 320Tyr?Val?Gly?Met?Asp?Ala?Leu?Asp?Met?Glu?Lys?Trp?Arg?Phe?Val?Ser
325 330 335Asp?Ser?Pro?Gly?Thr?Ser?Val?Ala?Val?Ser?Ala?Val?Leu?Met?Gly?Leu
340 345 350Val?Met?Val?Gly?Arg?Ala?Ala?Phe?Val?Phe?Pro?Leu?Ser?Phe?Leu?Ser
355 360 365Asn?Leu?Ala?Lys?Lys?Ser?Thr?Ser?Glu?Lys?Ile?Ser?Phe?Arg?Glu?Gln
370 375 380Ile?Ile?Ile?Trp?Trp?Ala?Gly?Leu?Met?Arg?Gly?Ala?Val?Ser?Met?Ala385 390 395 400Leu?Ala?Tyr?Asn?Gln?Phe?Thr?Arg?Gly?Gly?His?Thr?Gln?Leu?Arg?Gly
405 410 415Asn?Ala?Ile?Met?Ile?Thr?Ser?Thr?Ile?Thr?Ile?Val?Leu?Phe?Ser?Thr
420 425 430Val?Val?Phe?Gly?Leu?Met?Thr?Lys?Pro?Leu?Ile?Arg?Phe?Leu?Leu?Pro
435 440 445His?Pro?Lys?Pro?Thr?Ala?Ser?Met?Leu?Ser?Asp?Gln?Ser?Thr?Pro?Lys
450 455 460Ser?Met?Glu?Ala?Pro?Phe?Leu?Gly?Ser?Gly?Gln?Asp?Ser?Phe?Asp?Asp465 470 475 480Ser?Leu?Ile?Gly?Val?His?Arg?Pro?Asn?Ser?Ile?Arg?Ala?Leu?Leu?Thr
485 490 495Thr?Pro?Ala?His?Thr?Val?His?Tyr?Tyr?Trp?Arg?Lys?Phe?Asp?Asn?Ala
500 505 510Phe?Met?Arg?Pro?Met?Phe?Gly?Gly?Arg?Gly?Phe?Val?Pro?Phe?Val?Pro
515 520 525Gly?Ser?Pro?Thr?Glu?Arg?Ser?Glu?Pro?Asn?Leu?Pro?Gln?Trp?Gln
530 535 540
Claims (3)
1. cotton Na
+/ H
+Antiport protein gene GhNHX1 is characterized in that having the nucleotide sequence shown in the SEQ IN NO 1, the aminoacid sequence shown in the SEQ IN NO 2:
(1) information of SEQ IN NO 1
(a) sequence signature:
*Length: 9.485 base pairs
*Type: nucleic acid
*Chain: two strands
*Topological framework: linearity
(b) molecule type: cDNA
(c) suppose: not
(d) antisense: not
(e) initial source: cotton
(f) sequence description: SEQ IN NO.1
ACGCGGGGCAACACAGTCTTGATTTTGATCGTTTTTCGCTCCCATCGAAAGCGAAGATTT 60
TAAGCTGAAAAAAGAAGAGAGGAAAATTGTGGCAATTTGTTGGTGAGAAAGTCGAAGATT 120
CACGTGGGTAAGCTCCATAAACAGTGAAACATTGGATTTTCTTTTTTGTTTTTGTTTTCT 180
CAAGCTCTCTCTTCGAATTTACTCGTCTCTTTGAAACTGTCCGTTTTTTTTTGGTTCAAT 240
AAAATCGCAAATTATTTGCTAATTTAGAGAAGAAAATTGAACGGAGCTGAAACAAGGATG 300
ATTTGTTGCTGCATGATGTTGATTCTCCAAAACGATTCGAGTGCTTAAGGATTTTAAGAT 360
TAGAAAGTTCTTGAAATGGACAGTTCAGAGGCATAAAAATTTTCGAAGATTTACATTGTT 420
GAAGGAGAGCTTAAATCTGAAGCCTTGGACTACAACTGTTTCAGTTAGAAGGAATTGGTG 480
TTTAATAAAATTTGATTTAAAAAGAGGTCAATATGGTGGCTCCGCAGTTAGCTGCTGTCT 540
TTACTAAGTTGCAGACACTATCTACTTCAGACCATGCGTCTGTGGTCTCCATGAACATAT 600
TTGTAGCGCTTCTTTGTGCTTGCATTGTGATTGGTCATCTTTTGGAGGAGAATAGATGGA 660
TGAACGAATCAATTACTGCCCTTATCATTGGTGTTTTTACTGGGGTCATTATTTTGTTGA 720
CAAGTGGGGGTAAAAGCTCTCATCTTTTAGTCTTCAGTGAAGATCTGTTCTTTATCTATC 780
TTCTGCCCCCTATTATATTCAATGCTGGGTTTCAGGTGAAAAAGAAGCAATTTTTCCGTA 840
ACTTTATCACCATCATGCTGTTTGGGGCTGTTGGTACACTAATATCTTGTACAATTATCT 900
CTTTAGGTGTAATTAACTTCTTCAAGGAAATGGACATTGGCTCCTTAGACATTGGAGATT 960
TTCTAGCAATTGGTGCAATATTTGCTGCGACAGATTCTGTTTGCACACTGCAGGTGCTTA 1020
ATCAGGATGAGACTCCATTACTCTACAGTTTGGTTTTCGGAGAGGGTGTTGTAAATGATG 1080
CAACATCTGTGGTGCTTTTCAATGCAATCCAGAGTTTTGACCTCGTTAATACCAGTCCTA 1140
GAATTCTTCTGGAGTTTATTGGCAGCTTTTTGTATTTATTTTTAGCAAGCACTATGCTGG 1200
GAGTGATTGTTGGGTTGGTTAGTGCTTACATCATCAAAAAGTTGTACTTTGGAAGGCACT 1260
CAACAGATCGTGAATTTGCTCTTATGATGCTTATGGCATACCTTTCGTATATCATGGCTG 1320
AACTGTTCTATTTGAGTGGCATTCTTACAGTATTCTTTTGTGGGATTGTGATGTCACATT 1380
ATACCTGGCACAATGTAACTGAGAGTTCAAGAGTAACTACAAAGCATGCCTTTGCTACCT 1440
TGTCATTTGTTGCTGAGACTTTTCTCTTTCTTTATGTCGGGATGGATGCTTTGGACATGG 1500
AGAAGTGGAGATTTGTCAGTGATAGCCCTGGAACGTCAGTTGCTGTTAGTGCTGTGCTGA 1560
TGGGTCTTGTTATGGTTGGAAGAGCGGCTTTTGTGTTTCCCCTGTCATTTTTATCCAACT 1620
TGGCAAAGAAATCAACTAGTGAGAAAATCAGCTTCAGGGAACAAATTATAATATGGTGGG 1680
CTGGGCTCATGAGAGGCGCTGTATCTATGGCACTTGCATATAATCAGTTTACAAGGGGGG 1740
GCCATACTCAGTTGCGAGGAAATGCAATTATGATTACAAGCACCATAACCATTGTTCTAT 1800
TCAGCACTGTGGTTTTTGGTTTAATGACTAAACCTCTAATAAGGTTCTTGCTGCCTCATC 1860
CCAAACCAACAGCCAGCATGCTCTCAGACCAATCCACTCCAAAATCAATGGAGGCACCAT 1920
TTCTCGGAAGCGGCCAGGACTCTTTTGATGATAGTTTAATTGGAGTTCATCGACCAAACA 1880
GCATTCGTGCACTTCTTACAACTCCAGCACACACTGTTCATTACTATTGGCGAAAGTTTG 2040
ATAATGCCTTCATGCGCCCTATGTTTGGTGGCCGGGGTTTTGTGCCCTTCGTTCCTGGCT 2100
CCCCAACAGAAAGGAGTGAACCTAATCTGCCTCAATGGCAATGAGGTGGTTGAACAAGAT 2160
CTCTACAAAAATGTACATGTAATATAACAATGCAGTCGGTTGCAAAAAACATGCTTCTGG 2220
CGAGAAGCCAGTGCGGTATGCTTTGTATGTTTCATGTATAGGCTATATTTTGTTGGTTTT 2280
CAAGTTTCCTCAAGAGGTTCTTGTTTATTCTCCCCGAAACTACCTTCGCACCTGATGCTA 2340
TCTTTCCATTTGACATTTACGAATATTTATGATCTGGGTGAAGCTTAGGGGTAGGTGTGC 2400
CATTCTATTTTGTACGTATACGAGTATTTATTTTGTGTTTATATCAGTGTGTTTAGTTTT 2460
TATTTTTATTAAAAAAAAAAAAAAA?2485
(2) information of SEQ IN NO.2
(a) sequence signature
*Length: 543 amino acid
*Type: amino acid
*Chain: strand
*Topological framework: linearity
(b) molecule type: protein
(c) sequence description: SEQ IN NO.2MVAPQLAAVFTKLQTLSTSDHASVVSMNIFVALLCACIVIGHLLEENRWMNE SITALIIGVFTGVIILLTSGGKSSHLLV 81FSEDLFFIYLLPPIIFNAGFQVKKKQFFRNFITIMLFGAVGTLISCTIISLGVI NFFKEMDIGSLDIGDFLAIGAIFAAT 161DSVCTLQVLNQDETPLLYSLVFGEGVVNDATSVVLFNAIQSFDLVNTSPRILL EFIGSFLYLFLASTMLGVIVGLVSAY 241IKKLYFGRHSTDREFALMMLMAYLSYIMAELFYLSGILTVFFCGIVMSHYTWH NVTESSRVTTKHAFATLSFVAETFLFL 321YVGMDALDMEKWRFVSDSPGTSVAVSAVLMGLVMVGRAAFVFPLSFLSNLAKK STSEKISFREQIIIWWAGLMRGAVSMA 401LAYNQFTRGGHTQLRGNAIMITSTITIVLFSTVVFGLMTKPLIRFLLPHPKPT ASMLSDQSTPKSMEAPFLGSGQDSFDD 461SLIGVHRPNSIRALLTTPAHTVHYYWRKFDNAFMRPMFGGRGFVPFVPGSPTE RSEPNLPQWQ 543
2. Na according to claim 1
+/ H
+The cloning process of antiport protein gene GhNHX1 is characterized in that extracting total RNA from cotton leaf, utilizes the total RNA reverse transcription of 2 micrograms to become cDNA then, according to the Na in other plant
+/ H
+The conservative aminoacid sequence of antiport albumen designs a pair of degenerate primer:
Forward primer: 5 '-CC (GATC) CC (GATC) AT (TAC) AT (TAC) TT (TC) AA (TC) GC-3 '
Reverse primer: 5 '-GT (GATC) AC (GATC) TT (AG) TGCCA (GATC) GT (AG) TA-3 '
Carry out conventional polymerase chain reaction, the dna fragmentation that amplifies is connected on the pGEM-T carrier, transform DH5 α cell, be used for sequencing, the cDNA that obtains total length through 3 ' and 5 ' terminal rapid amplifying is 2485bp then.
3. the application of 1 described GhNHX1 resistant gene of salt as requested is characterized in that this gene overexpression in tobacco, can improve the salt tolerance of plant, and this gene can be used for the genetic transformation of other single dicotyledonss, improves its salt tolerance.
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|---|---|---|---|
| CNB021515301A CN1190492C (en) | 2002-12-31 | 2002-12-31 | Cotton Na+/H+ reverse transport protein gene and its cloning method and use |
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|---|---|---|---|
| CNB021515301A CN1190492C (en) | 2002-12-31 | 2002-12-31 | Cotton Na+/H+ reverse transport protein gene and its cloning method and use |
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| Publication Number | Publication Date |
|---|---|
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| CN1190492C CN1190492C (en) | 2005-02-23 |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100355896C (en) * | 2004-07-30 | 2007-12-19 | 中国科学院上海生命科学研究院 | Method for breeding halophyte of double transgene |
| CN106319082A (en) * | 2016-11-09 | 2017-01-11 | 中国农业科学院棉花研究所 | Method for identifying cotton seedling-stage salt tolerance |
| CN106868039A (en) * | 2017-03-02 | 2017-06-20 | 中国农业科学院棉花研究所 | A kind of expression vector and its application in genetically modified plants are cultivated |
| CN109105235A (en) * | 2018-08-17 | 2019-01-01 | 河北省农林科学院昌黎果树研究所 | A kind of identification method of grape rootstock salt tolerance |
| CN112322629A (en) * | 2020-10-13 | 2021-02-05 | 河南农业大学 | Application of gene GhNHX4A in aspect of salt tolerance of plants |
-
2002
- 2002-12-31 CN CNB021515301A patent/CN1190492C/en not_active Expired - Fee Related
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100355896C (en) * | 2004-07-30 | 2007-12-19 | 中国科学院上海生命科学研究院 | Method for breeding halophyte of double transgene |
| CN106319082A (en) * | 2016-11-09 | 2017-01-11 | 中国农业科学院棉花研究所 | Method for identifying cotton seedling-stage salt tolerance |
| CN106868039A (en) * | 2017-03-02 | 2017-06-20 | 中国农业科学院棉花研究所 | A kind of expression vector and its application in genetically modified plants are cultivated |
| CN106868039B (en) * | 2017-03-02 | 2019-11-29 | 中国农业科学院棉花研究所 | A kind of expression vector and its application in cultivation genetically modified plants |
| CN109105235A (en) * | 2018-08-17 | 2019-01-01 | 河北省农林科学院昌黎果树研究所 | A kind of identification method of grape rootstock salt tolerance |
| CN112322629A (en) * | 2020-10-13 | 2021-02-05 | 河南农业大学 | Application of gene GhNHX4A in aspect of salt tolerance of plants |
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| CN1190492C (en) | 2005-02-23 |
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