CN1706951A - Tonoplast Membrane Na+/H+ Antiporter 2 Gene and Its Application - Google Patents
Tonoplast Membrane Na+/H+ Antiporter 2 Gene and Its Application Download PDFInfo
- Publication number
- CN1706951A CN1706951A CN 200510043511 CN200510043511A CN1706951A CN 1706951 A CN1706951 A CN 1706951A CN 200510043511 CN200510043511 CN 200510043511 CN 200510043511 A CN200510043511 A CN 200510043511A CN 1706951 A CN1706951 A CN 1706951A
- Authority
- CN
- China
- Prior art keywords
- iris
- sword
- transport protein
- reverse transport
- vacuolar membrane
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 239000012528 membrane Substances 0.000 title claims abstract description 61
- 101150028074 2 gene Proteins 0.000 title description 8
- 102000003669 Antiporters Human genes 0.000 title description 2
- 108090000084 Antiporters Proteins 0.000 title description 2
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 75
- 238000000034 method Methods 0.000 claims abstract description 23
- 230000014509 gene expression Effects 0.000 claims abstract description 7
- 238000011160 research Methods 0.000 claims abstract description 6
- 230000009465 prokaryotic expression Effects 0.000 claims abstract description 5
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 3
- 108010078791 Carrier Proteins Proteins 0.000 claims description 70
- 102000014914 Carrier Proteins Human genes 0.000 claims description 70
- 241000196324 Embryophyta Species 0.000 claims description 36
- 239000013612 plasmid Substances 0.000 claims description 25
- 239000013598 vector Substances 0.000 claims description 17
- 230000009261 transgenic effect Effects 0.000 claims description 16
- 241000589158 Agrobacterium Species 0.000 claims description 12
- 230000008859 change Effects 0.000 claims description 10
- 238000006243 chemical reaction Methods 0.000 claims description 10
- 101150066002 GFP gene Proteins 0.000 claims description 9
- 241000209140 Triticum Species 0.000 claims description 7
- 235000021307 Triticum Nutrition 0.000 claims description 7
- 108090000848 Ubiquitin Proteins 0.000 claims description 6
- 102000044159 Ubiquitin Human genes 0.000 claims description 6
- 239000005090 green fluorescent protein Substances 0.000 claims description 5
- 101150073246 AGL1 gene Proteins 0.000 claims description 4
- 239000002585 base Substances 0.000 claims description 4
- 230000008676 import Effects 0.000 claims description 4
- 230000001404 mediated effect Effects 0.000 claims description 4
- 240000008042 Zea mays Species 0.000 claims description 3
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 3
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 3
- 239000003513 alkali Substances 0.000 claims description 3
- 235000005822 corn Nutrition 0.000 claims description 3
- 230000004927 fusion Effects 0.000 claims description 3
- 241000193830 Bacillus <bacterium> Species 0.000 claims description 2
- 241000206602 Eukaryota Species 0.000 claims description 2
- 108010043121 Green Fluorescent Proteins Proteins 0.000 claims description 2
- 102000004144 Green Fluorescent Proteins Human genes 0.000 claims description 2
- 101100288095 Klebsiella pneumoniae neo gene Proteins 0.000 claims description 2
- 108090000364 Ligases Proteins 0.000 claims description 2
- 108060008682 Tumor Necrosis Factor Proteins 0.000 claims description 2
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 claims description 2
- 230000001939 inductive effect Effects 0.000 claims description 2
- JEIPFZHSYJVQDO-UHFFFAOYSA-N iron(III) oxide Inorganic materials O=[Fe]O[Fe]=O JEIPFZHSYJVQDO-UHFFFAOYSA-N 0.000 claims description 2
- 239000003550 marker Substances 0.000 claims description 2
- 238000000520 microinjection Methods 0.000 claims description 2
- 239000002245 particle Substances 0.000 claims description 2
- 230000009466 transformation Effects 0.000 claims description 2
- 150000003839 salts Chemical class 0.000 abstract description 15
- 230000002337 anti-port Effects 0.000 abstract description 8
- 238000010367 cloning Methods 0.000 abstract description 5
- 239000013604 expression vector Substances 0.000 abstract description 3
- 102000004169 proteins and genes Human genes 0.000 abstract 2
- 108091028043 Nucleic acid sequence Proteins 0.000 abstract 1
- 239000000427 antigen Substances 0.000 abstract 1
- 102000036639 antigens Human genes 0.000 abstract 1
- 108091007433 antigens Proteins 0.000 abstract 1
- 150000007523 nucleic acids Chemical group 0.000 abstract 1
- 210000001236 prokaryotic cell Anatomy 0.000 abstract 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 8
- 230000003321 amplification Effects 0.000 description 8
- 230000004087 circulation Effects 0.000 description 8
- 238000003199 nucleic acid amplification method Methods 0.000 description 8
- 210000003934 vacuole Anatomy 0.000 description 8
- 241000894006 Bacteria Species 0.000 description 7
- 108090000790 Enzymes Proteins 0.000 description 7
- 102000004190 Enzymes Human genes 0.000 description 7
- 238000009395 breeding Methods 0.000 description 7
- 210000004027 cell Anatomy 0.000 description 6
- 239000012634 fragment Substances 0.000 description 6
- 230000001488 breeding effect Effects 0.000 description 5
- 239000002299 complementary DNA Substances 0.000 description 5
- 238000012163 sequencing technique Methods 0.000 description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- 241001302160 Escherichia coli str. K-12 substr. DH10B Species 0.000 description 4
- 230000000968 intestinal effect Effects 0.000 description 4
- 238000004153 renaturation Methods 0.000 description 4
- 230000004044 response Effects 0.000 description 4
- 239000011780 sodium chloride Substances 0.000 description 4
- 230000004083 survival effect Effects 0.000 description 4
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 238000010353 genetic engineering Methods 0.000 description 3
- 238000010839 reverse transcription Methods 0.000 description 3
- 235000016068 Berberis vulgaris Nutrition 0.000 description 2
- 241000335053 Beta vulgaris Species 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- 238000002105 Southern blotting Methods 0.000 description 2
- 108700019146 Transgenes Proteins 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 238000009396 hybridization Methods 0.000 description 2
- 230000015784 hyperosmotic salinity response Effects 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 108090000915 Aminopeptidases Proteins 0.000 description 1
- 244000098360 Atriplex halimus Species 0.000 description 1
- 235000005482 Atriplex halimus Nutrition 0.000 description 1
- 235000021537 Beetroot Nutrition 0.000 description 1
- 241000701489 Cauliflower mosaic virus Species 0.000 description 1
- 229920000742 Cotton Polymers 0.000 description 1
- 241000195493 Cryptophyta Species 0.000 description 1
- 101100175482 Glycine max CG-3 gene Proteins 0.000 description 1
- 244000299507 Gossypium hirsutum Species 0.000 description 1
- 244000020551 Helianthus annuus Species 0.000 description 1
- 235000003222 Helianthus annuus Nutrition 0.000 description 1
- 240000005979 Hordeum vulgare Species 0.000 description 1
- 235000007340 Hordeum vulgare Nutrition 0.000 description 1
- 235000007688 Lycopersicon esculentum Nutrition 0.000 description 1
- 244000021685 Mesembryanthemum crystallinum Species 0.000 description 1
- 240000001140 Mimosa pudica Species 0.000 description 1
- 235000016462 Mimosa pudica Nutrition 0.000 description 1
- 101150039148 NHX1 gene Proteins 0.000 description 1
- 235000000715 Sarcobatus vermiculatus Nutrition 0.000 description 1
- CWHJIJJSDGEHNS-MYLFLSLOSA-N Senegenin Chemical compound C1[C@H](O)[C@H](O)[C@@](C)(C(O)=O)[C@@H]2CC[C@@]3(C)C(CC[C@]4(CCC(C[C@H]44)(C)C)C(O)=O)=C4[C@@H](CCl)C[C@@H]3[C@]21C CWHJIJJSDGEHNS-MYLFLSLOSA-N 0.000 description 1
- 240000003768 Solanum lycopersicum Species 0.000 description 1
- 241000863480 Vinca Species 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000009056 active transport Effects 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 239000013599 cloning vector Substances 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 101150054900 gus gene Proteins 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 229960002523 mercuric chloride Drugs 0.000 description 1
- LWJROJCJINYWOX-UHFFFAOYSA-L mercury dichloride Chemical compound Cl[Hg]Cl LWJROJCJINYWOX-UHFFFAOYSA-L 0.000 description 1
- 230000000050 nutritive effect Effects 0.000 description 1
- 230000002018 overexpression Effects 0.000 description 1
- 239000002574 poison Substances 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 239000000523 sample Substances 0.000 description 1
- 238000012772 sequence design Methods 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 239000009871 tenuigenin Substances 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 230000002103 transcriptional effect Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 230000032258 transport Effects 0.000 description 1
- 230000007279 water homeostasis Effects 0.000 description 1
Images
Landscapes
- Peptides Or Proteins (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The present invention discloses the nucleic acid sequence and corresponding amino acid sequence of one kind of sword-like iris vacuolar membrane Na+/H+ antiport protein-2 gene, and the biotechnological application in improving salt resisting quality of crop and pasture. The sword-like iris vacuolar membrane Na+/H+ antiport protein-2 gene is one member in sword-like iris vacuolar membrane Na+/H+ antiport protein family and is related positively to the salt resisting property of sword-like iris. Based on the gene sequence, the gene may be cloned and prokaryotic expression vector may be constituted for expression in prokaryotic cell in great amount to obtain Na+/H+ antiport protein-2 for protein structure research, function research and antigen preparation. After cloning the gene, various kinds of eukaryotic expression vector may be constituted, and the gene may be led into crop and pasture through different methods to breed excellent salt tolerant variety.
Description
Technical field
The present invention relates to sword-like iris vacuolar membrane Na
+/ H
+ Reverse transport protein 2 genes (IINHX2) belong to biological technical field.
Technical background
Plant is eliminated the strategy that Na+ poisons and comprises: reduce Na+ absorption, Na+ efflux separating with Na+.It is the process of a complexity that Na+ absorbs.Na+ efflux and separating is indirect active transport process, mainly regulate by the Na+/H+ reverse transport protein.Na
+/ H
+Reverse transport protein (Na
+/ H
+Antiporter or exchanger, NHA or NHE) is ubiquitous a kind of translocator in the film system of bacterium, yeast, algae, animal and higher plant, participates in intracytoplasmic pH, Na
+Vital movements such as concentration adjustment and cell volume variation.At first since finding Na+/H+ antiport activity on the vacuole skin of beet root storage tissue, people find at halophytes such as saltbush, facultative CAM plants ice plant, beet etc. and before than the glycophyte of salt tolerant such as barley, beach car, ubiquity Na on the vacuole skins of Vinca, cotton, Sunflower Receptacle etc. in succession from Blumwald in 1985 and Plooe
+/ H
+The antiport activity.Nass etc. have also found a kind of Na on the vacuole skin (prevacuole) before zymic
+/ H
+Reverse transport protein.A large amount of experiments show Na
+/ H
+Antiport is active to be had or not with just closely related with plant and zymic salt tolerance, and under high salt concn plant and yeast can be respectively by the Na on plasma membrane and the vacuole skin
+/ H
+Antiport is with Na
+Transport cell and with Na
+Compartmentation is kept Na in the tenuigenin in vacuole
+Stable state and Na
+/ K
+Than relatively stable, to adapt to the salt marsh environment.Salt-tolerant plant can by the Na+/H+ reverse transport protein with most of Na+ compartmentation in vacuole, thereby can avoid ion to coerce, can carry out osmoregulation again.So Na
+/ H
+The effect of reverse transport protein in plant salt endurance more and more comes into one's own.People have obtained coding Na in succession from bacterium, yeast, animal and higher plant at present
+/ H
+The gene of reverse transport protein.
(as the AtNHX1 of Arabidopis thaliana and zymic NHX1 etc.) are transformed in the salt sensitive plant the gene of Na+/H+ reverse transport protein on the coding vacuole skin, and to make its overexpression on vacuole skin be feasible plant genetic engineering strategy with the raising plant salt endurance.Obtained the transfer-gen plant of high salt tolerant plants such as Arabidopis thaliana, tomato, beets.
Chinese small iris is a kind of halophytes, and the most outstanding characteristics of Chinese small iris are exactly to plant on the geavy salt alkaline earth that other high-yield crops can not grow.The gene of Na+/H+ reverse transport protein 2 and the application in the transgenic plant genetic engineering breeding thereof are not also reported on the at present relevant sword-like iris vacuolar membrane.
Summary of the invention
At also not reporting the Chinese small iris NHX gene of salt tolerant alkali and the present situation that in plant transgene breeding engineering, is applied thereof at present, the invention provides a kind of sword-like iris vacuolar membrane Na
+/ H
+ Reverse transport protein 2 genes, recombinant vectors and plant transgene breeding method are with sword-like iris vacuolar membrane Na
+/ H
+ Reverse transport protein 2 is gene constructed on the conversion plasmid of expression of plants, and transformed wheat obtains transfer-gen plant.
Sword-like iris vacuolar membrane Na provided by the present invention
+/ H
+ Reverse transport protein 2 genes (IINHX2) have the sequence shown in SEQ IDNO.1:
ATGGGGTCTG??GTATGGAGGA??TCTGCTGGTG??AGGCTGGGTG??TCGTGTCGTC??CACCTCCGAC?????60
CATGCCTCCG??TGGTCTCCAT??CAACCTCTTC??GTAGCTCTCC??TCTGCGCATG??CATAGTCATC????120
GGACACCTCC??TGGAGGAGAA??TAGATGGATG??AACGAGTCCA??TCACCGCTCT??AGCCATCGGG????180
TTGTGCACTG??GCGTTGTGAT??TCTTCTGACG??ACAAATGGGA??AGAGCTCTCA??TATATTCGTA????240
TTCAGCGAAG??ATCTCTTCTT??CATCTACCTC??CTCCCTCCAA??TCATCTTCAA??CGCCGGGTTT????300
CAAGTAAAAA??AGAAACAATT??TTTCCGCAAC??TTCATGACAA??TTATGCTGTT??TGGTGCAGTT????360
GGGACCCTCA??TCTCCTTTGT??CATAATTTCT??CTTGGTGCAA??TTGGTTTATT??CAGAAAAATG????420
AACATAGGTC?CACTGGAAGT?TGGAGATTTT?CTTGCAATTG?GGGCAATCTT?TTCTGCAACA??480
GATTCTGTTT?GCACCTTGCA?GATTCTTAAC?CAGGATGAAA?CACCATTATT?ATATAGCTTG??540
GTCTTTGGTG?AGGGAGTTGT?CAATGATGCC?ACATCAGTGG?TGCTTTTCAA?CGCAATACAA??600
AACTTCGATC?TTGAACATAT?TGATGCAGGC?ATTGCAGCAA?AATTTATTGG?AAATTTCTTT??660
TATTTATTTG?TCAGCAGCAC?CTTCTTGGGA?GCATTTGCTG?GATTGCTTAG?TGCGTATATC??720
ATAAAAAAGT?TGTATTATGG?AAGGCATTCT?ACTGATCGTG?AAGTTGCACT?CATGATGCTT??780
ATGGCGTACC?TTTCATATAT?GCTGGCTGAG?CTGTTAGATC?TAAGTGGTAT?CCTCACGGTT??840
TTCTTCTGCG?GCATAGTAAT?GTCCCATTAT?ACGTGGCACA?ATGTGACAGA?GAGTTCGAGA??900
GTCACAACCA?AGCATGCTTT?TGCAACCTTG?TCATTTATTT?CGGAGATATT?CCTCTTCCTT??960
TATGTTGGCA?TGGATGCATT?AGACATTGAA?AAGTGGAGAT?TTGTCAGCGA?CAGTCCTGGG??1020
AAATCAATTG?GTGTTAGCGC?AATCATAATT?GGTTTAGTTT?TGATTGGAAG?AGCAGCTTTC??1080
GTCTTCCCGC?TGTCTTTCCT?ATCCAACTTA?GCTAAGAAGT?CCCCTAATGA?AAAAATCACC??1140
TTCAACCAGC?AAGTTACCAT?ATGGTGGGCA?GGTTTGATGA?GAGGTGCTGT?ATCAATTGCA??1200
CTTGCTTACA?ATCAGTTCAC?AAGAGCTGGT?CATACTCAAC?TTCCAGGGAA?TGCAATGATG??1260
ATCACCAGTA?CCATCACAGT?TGTGCTATTT?AGCACTGTGG?TATTCGGATT?ATTGACAAAG??1320
CCTTTGATAA?GGCTCCTGTT?GCCTCCTAGT?ACAAAGCATC?TTACCGGCAG?TTGCAGTTTC??1380
AACTCTGAGC?CCTCTACCCC?AAAATTTTA?TCCGCTGCAC?TACTAGAAAA?TATTCCAGGA???1440
TTTGAAGCAG?AAGGAGGAAC?AACAAACCTT?GGGGCTCGGC?CTTCAAGCCT?TCGAATGCTC??1500
CTTATGAAGC?CAACTCACAC?GGTTCATTAC?TATTGGCGCA?AGTTTGATGA?TGCTTTCATG??1560
CGTCCAATGT?TTGGAGGGCG?AGGCTTTGTT?CCTTTTGTTC?CCGGTTCACC?TACTGAACAA??1620
AGTGTTCATG?GATGGGAATG?A????????????????????????????????????????????1641
Wherein, comprise with sequence shown in the SEQ ID NO.1 having the sequence of at least 95% homogeny.
Sword-like iris vacuolar membrane Na
+/ H
+ Reverse transport protein 2 genes (IINHX2) aminoacid sequence is shown in sequence table SEQ IDNO.2:
MGSGMEDLLV?RLGVVSSTSD?HASVVSINLF?VALLCACIVI?GHLLEENRWM?NESITALAIG???60
LCTGVVILLT?TNGKSSHIFV?FSEDLFFIYL?LPPIIFNAGF?QVKKKQFFRN?FMTIMLFGAV???120
GTLISFVIIS?LGAIGLFRKM?NIGPLEVGDF?LAIGAIFSAT?DSVCTLQILN?QDETPLLYSL???180
VFGEGVVNDA?TSVVLFNAIQ?NFDLEHIDAG?IAAKFIGNFF?YLFVSSTFLG?AFAGLLSAYI???240
IKKLYYGRHS?TDREVALMML?MAYLSYMLAE?LLDLSGILTV?FFCGIVMSHY?TWHNVTESSR???300
VTTKHAFATL?SFISEIFLFL?YVGMDALDIE?KWRFVSDSPG?KSIGVSAIII?GLVLIGRAAF???360
VFPLSFLSNL?AKKSPNEKIT?FNQQVTIWWA?GLMRGAVSIA?LAYNQFTRAG?HTQLPGNAMM???420
ITSTITVVLF?STVVFGLLTK?PLIRLLLPPS?TKHLTGSCSF?NSEPSTPKFL?SAALLENIPG???480
FEAEGGTTNL?GARPSSLRML?LMKPTHTVHY?YWRKFDDAFM?RPMFGGRGFV?PFVPGSPTEQ???540
SVHGWE??????????????????????????????????????????????????????????????546
Wherein, comprise with sequence shown in the SEQ ID NO.2 having the sequence of at least 95% homogeny.
Above-mentioned sword-like iris vacuolar membrane Na
+/ H
+ Reverse transport protein 2 genes can be building up in the prokaryotic expression carrier, by research method well known in the art with sword-like iris vacuolar membrane Na
+/ H
+ Reverse transport protein 2 genes import prokaryotic organism, and by inducing, great expression goes out sword-like iris vacuolar membrane Na
+/ H
+Reverse transport protein 2.Wherein, the preferred prokaryotic expression carrier pET-24a of prokaryotic expression carrier.
Described sword-like iris vacuolar membrane Na
+/ H
+ Reverse transport protein 2 can be used for studying sword-like iris vacuolar membrane Na
+/ H
+The structure of reverse transport protein 2, function and Antibody Preparation.
Described sword-like iris vacuolar membrane Na
+/ H
+ Reverse transport protein 2 genes can pass through microinjection, particle gun, agriculture bacillus mediated, pollen tube channel method with sword-like iris vacuolar membrane Na
+/ H
+ Reverse transport protein 2 genes change crop and herbage over to, cultivate the colory kind of anti-salt/be.
Described sword-like iris vacuolar membrane Na
+/ H
+ Reverse transport protein 2 genes can be building up to any based in the Ti-plasmid binary vector, by research method well known in the art with sword-like iris vacuolar membrane Na
+/ H
+ Reverse transport protein 2 genes import in the Agrobacterium, carry out Agrobacterium-mediated Transformation.
Described sword-like iris vacuolar membrane Na
+/ H
+ Reverse transport protein 2 genes are structured on the Ti-plasmid binary vector pBIUbiGFP of expression of plants as goal gene; The T-DNA25bp tumor-necrosis factor glycoproteins that LB and RB are wherein arranged, the promotor P-nos of rouge alkali synthetase gene and terminator T-nos also have the nptII that uses as selective marker in eukaryote, and the microbiotic that is used to select is kantlex and G418; In pBIUbiGFP, also have corn ubiquitin super promoter Ubi and green fluorescent protein GFP gene; Sword-like iris vacuolar membrane Na
+/ H
+ Reverse transport protein 2 genes are inserted on the Ubiquitin promotor and the multiple clone site between the GFP gene of pBIUbiGFP plasmid, and form fusion gene with the GFP gene, express a N-terminal and are connected with the proteic sword-like iris vacuolar membrane Na of GFP
+/ H
+ Reverse transport protein 2.
Sword-like iris vacuolar membrane Na provided by the present invention
+/ H
+The Ti-plasmid binary vector transgenic breeding method of reverse transport protein 2 genes is:
1) the double base conversion transfer vector plasmid pBIUbiGFP:IINHX2 that makes up transforms in Agrobacterium, infects plant by Agrobacterium shoot apex conversion method, thereby obtains transfer-gen plant.Preferred double base transforms recombinant vectors pBIUbiGFP:IINHX2 and transforms in Agrobacterium AGL1, infects wheat by Agrobacterium shoot apex conversion method, thereby obtains transgenic wheat.Wherein, crown gall soil Agrobacterium AGL1 is public bacterial strain.
2) detection method of transgenic line: detect IINHX2 gene in the rotaring gene plant blade with PCR and SOUTHERN blot hybridization method.
The invention provides a kind of sword-like iris vacuolar membrane Na
+/ H
+ Reverse transport protein 2 genes and application thereof, broken through the present situation that the IINHX2 gene also is not applied in the transgenic plant genetic engineering breeding, first with the gene constructed conversion plasmid of IINHX2 at expression of plants, with Ti-plasmid binary vector pBIUbiGFP:IINHX2 transformed wheat and obtained transfer-gen plant, the salt tolerance through identifying transfer-gen plant significantly is improved.The T of IINHX2 gene transgenic wheat
1Show tangible salt resistance for plant, not the T of homophyletic system
1For the surviving rate of plant on 170mM NaCl is 66%-83%, is higher than contrast China spring (44%), and the surviving rate on 170mM NaCl is 27%-38%, is higher than contrast China spring (0%).
The bright transgenosis recombinant vectors of we can be used as the kind, strain of commercial use or directly uses on producing or carry out agro-ecology breeding and transgenic plant to improve the salt resistance of crop as genetic resources.
Description of drawings
Fig. 1, sword-like iris vacuolar membrane Na
+/ H
+ Reverse transport protein 2 gene Ti-plasmid binary vector design of graphics
Fig. 2, sword-like iris vacuolar membrane Na
+/ H
+ Reverse transport protein 2 gene degenerated primer amplifications
Wherein, 1,2 is respectively different template concentrations, and M is the two Maker that cut of the EcoRI+HindIII of λ DNA
Fig. 3, sword-like iris vacuolar membrane Na
+/ H
+3 ' RACE of reverse transport protein 2 genes
Wherein, M is 100bp Lader, the different repetition of 1,2,3 representatives
Fig. 4, sword-like iris vacuolar membrane Na
+/ H
+5 ' RACE of reverse transport protein 2 genes
Wherein, M is 100bp Lader, the different repetition of 1,2 representative
Fig. 5, sword-like iris vacuolar membrane Na
+/ H
+ Reverse transport protein 2 full length gene cDNA amplification
Wherein, M is 100bp Lader, the different repetition of 1,2 representative
Fig. 6, transgenosis T
1The Southern results of hybridization in generation
Wherein, M: λ DNA (HindIII+EcoRI), B53, B59: transfer-gen plant, wt: the wild-type China spring ,+: plasmid pBIUbiGFP:IINHX2
Embodiment
1. Chinese small iris Na
+/ H
+The pulsating clone of reverse transport protein 2 Gene Partial
The Trizol method is extracted the total RNA that sprouts the Chinese small iris seedling about 2 weeks, PowerScript
TMThe first chain cDNA is synthesized in the reversed transcriptive enzyme reverse transcription, use degenerate primer: P1:5 ｀-GAT TCT GTW TGC ACM YTR CAG GT-｀ 3, P2:5 ｀-CAT KAG MCC AGC CCA CCA WAT-｀ 3 carries out pcr amplification, obtains an amplification segment, cloning and sequencing.
According to following condition amplifying target genes fragment:
Reaction system: 2 * GC buffer I, 25 μ l, 10mM dNTP 3 μ l, each 1.5 μ l of 10 μ M primers 1 and primer 2, dna profiling 250ng, LA GC Taq enzyme 1 μ l adds water and supplies 50 μ l.
Response procedures is: 94 ℃ of pre-sex change 3min, carry out 36 circulations then, and each circulation comprises 94 ℃ of sex change 40s, 59 ℃ of renaturation 1min, 72 ℃ are extended 1min.Remain on 72 ℃ at last and extend 10min down.
2. Chinese small iris Na
+/ H
+The acquisition of reverse transport protein 2 genes 3 ｀ ends
According to Chinese small iris Na
+/ H
+ Reverse transport protein 2 Gene Partial fragments sequence, design 3 ' RACE gene specific primer P1:5 '-ACA ATG TGA CAG AGA GTT CG-3 ', nested primer P2:5 '-CTT ATGAAG CCA ACT CAC ACG-3 '.Use primer P1 and primer Olig (dT) earlier
18CDNA with reverse transcription is a template amplification, and 50 times of product dilutions are with nested primer P2 and Olig (dT)
18Through nest-type PRC, obtain an amplification segment, cloning and sequencing.
According to following condition amplifying target genes fragment:
Reaction system: 2 * GC buffer I, 25 μ l, 10mM dNTP 3 μ l, each 1.5 μ l of 10 μ M primers 1 and primer 2, DNA Template 250ng, LA GC Taq enzyme 1 μ l adds water and supplies 50 μ l.
Response procedures is: 94 ℃ of pre-sex change 3min, carry out 36 circulations then, and each circulation comprises 94 ℃ of sex change 40s, 55 ℃ of renaturation 1min, 72 ℃ are extended 2min.Remain on 72 ℃ at last and extend 10min down.
3. Chinese small iris Na
+/ H
+The acquisition of reverse transport protein 2 genes 5 ｀ ends
According to Chinese small iris Na
+/ H
+ Reverse transport protein 2 Gene Partial fragments sequence design 5 ' RACE gene specific primer .P1:5 '-GAA AGA CAG CGG GAA GAC GAA AG-3 ', nested primer P2:5 '-CCAACA TAA AGG AAG AGG AAT-3 '.Using earlier primer P1 and primer UMP, is template amplification with the cDNA of reverse transcription, and 50 times of product dilutions through nest-type PRC, obtain an amplification segment, cloning and sequencing with primer nested primer P2 and NUMP.
According to following condition amplifying target genes fragment:
Reaction system: 2 * GC buffer I, 25 μ l, 10mM dNTP 3 μ l, each 1.5 μ l of 10 μ M primers 1 and primer 2, DNA Template 250ng, LA GC Taq enzyme 1 μ l adds water and supplies 50 μ l.
Response procedures is: 94 ℃ of pre-sex change 3min, carry out 36 circulations then, and each circulation comprises 94 ℃ of sex change 40s, 55 ℃ of renaturation 1min, 72 ℃ are extended 3min.Remain on 72 ℃ at last and extend 10min down.
4. Chinese small iris Na
+/ H
+The acquisition of reverse transport protein 2 full length gene cDNA
Result according to 5 ｀ Race and 3 ｀ Race, designed primer to P1:5 '-ACC GGT ATG GGG TCTGGT ATG GA-3 ', P2:5 '-CCA TGG ATT CCC ATC CAT GAA CAC-3 ', amplification cDNA template obtains total length segment, cloning and sequencing.
According to following condition amplifying target genes fragment:
Reaction system: 2 * GC buffer I, 25 μ l, 10mM dNTP 3 μ l, each 1.5 μ l of 10 μ M primers 1 and primer 2, DNA Template 250ng, LA GC Taq enzyme 1 μ l adds water and supplies 50 μ l.
Response procedures is: 94 ℃ of pre-sex change 3min, carry out 36 circulations then, and each circulation comprises 94 ℃ of sex change 40s, 51 ℃ of renaturation 1min, 72 ℃ are extended 2min.Remain on 72 ℃ at last and extend 10min down.
5. sword-like iris vacuolar membrane Na+/H+ reverse transport protein 2 gene sequencing make up with cloning vector
The band that separates corresponding size behind the pcr amplification product electrophoresis is reclaimed the back to be connected with the pUCm-T carrier, transformed into escherichia coli DH10B competent cell, and by PCR method, digested plasmid method etc. verify clone product whether correct, check order then the proof this gene the success the form with cloned plasmids be stored among the intestinal bacteria DH10B.
6. the Ti-plasmid binary vector of sword-like iris vacuolar membrane Na+/H+ reverse transport protein 2 genes makes up
The plasmid that is used to make up double base conversion-expression vector is to be transformed by pBI121.At first the gus gene in the pBI121 is removed with BamH I and Sac I, be connected with the GFP gene that Sac I enzyme is cut with BamH I then with on the pIRES-EGFP plasmid, transformed into escherichia coli DH10B competent cell, and verify that by digested plasmid method etc. this novel vector (called after pBIGFP) is stored among the intestinal bacteria DH10B with correct form.
Promotor 35S (p35S) with the cauliflower mosaic virus in the carrier pBIGFP, remove with HindIII and BamH I, be connected with the corn ubiquitin super promoter Ubi that BamH I enzyme is cut with HindIII then with on the pAL76 plasmid, transformed into escherichia coli DH10B competent cell, and verify that by digested plasmid method etc. this novel vector (called after pBIUbiGFP) is stored among the intestinal bacteria DH10B with correct form.
Sword-like iris vacuolar membrane Na+/H+ reverse transport protein 2 genes that use as goal gene are inserted on the Ubiquitin promotor of pBIUbiGFP plasmid and the multiple clone site between the GFP gene and with the GFP gene and form fusion gene, express a N-terminal and be connected with the proteic sword-like iris vacuolar membrane Na+/H+ of GFP reverse transport protein 2, the restriction endonuclease of use is AgeI and NcoI.To connect product transformed into escherichia coli DH10B competent cell, and verify that by digested plasmid method etc. this novel vector (called after pBIUbiGFP:IINHX2) is stored among the intestinal bacteria DH10B with correct form.
7. the transgenic breeding of sword-like iris vacuolar membrane Na+/H+ reverse transport protein 2 gene Ti-plasmid binary vectors is got the seed of China spring, mercuric chloride sterilization 10min, and distilled water wash 3 times neatly places the 9cm culture dish, shows money or valuables one carries unintentionally under 25 ℃.At 4 ℃, vernalization is more than 1 month under the dark condition.Cut out vegetative point with double-edged razor blade, and drip the Agrobacterium AGL1 bacterium liquid that contains the pBIUbiGFP:IINHX2 plasmid that 1-2 drips, continue in culture dish, to be cultured to young leaves and grow, transplant.Filter out transfer-gen plant with PCR, transfer-gen plant is added generation.
Choose the T of two transgenic lines
1Generation, extract the total DNA that sprouts growing way blade in vigorous period, as probe, carry out Southern hybridization with sword-like iris vacuolar membrane Na+/H+ reverse transport protein 2 full length gene sequences, show that sword-like iris vacuolar membrane Na+/H+ reverse transport protein 2 genes exist in transgenic line B53 and B59 genome.
Choose the T of four transgenic lines
1Respectively after the MS nutritive medium of the NaCl that adds 170mM and 300mM is cultivated 5 days, whole transgenic lines shows the surviving rate higher than wild-type China spring for seedling.Wherein two transgenic lines reach 50% and 38% respectively in the surviving rate of 300mM, the death and the wild-type China spring is all wilted.(seeing Table 1)
Table 1: transgenic line T
1For the surviving rate under different salt concn relatively
| NaCl concentration | ????170mM? | ??300mM? | ||||
| Transgenic lines | The plant sum | Survival plant number | Survival rate | The plant sum | Survival plant number | Survival rate |
| China spring B22 B24 B53 B59 | ????16? ????15? ????16? ????19? ????18? | ????7? ????10? ????12? ????15? ????15? | ????44％? ????66％? ????69％? ????89％? ????83％? | ????16? ????15? ????16? ????19? ????18? | ????0? ????4? ????4? ????10? ????7? | ????0％? ????27％? ????25％? ????52％? ????38％? |
Sequence table
SEQ?ID?NO.1
＜110〉Shandong University
＜120〉sword-like iris vacuolar membrane Na
+/ H
+ Reverse transport protein 2 genes and application
<141>2005-5-13
<170>PatentIn?Version?2.1
<210>1
<211>1641
<212>DNA
＜213〉sword-like iris vacuolar membrane Na
+/ H
+ Reverse transport protein 2 genes
<400>
atggggtctg?gtatggagga?tctgctggtg?aggctgggtg?tcgtgtcgtc?cacctccgac????60
catgcctccg?tggtctccat?caacctcttc?gtagctctcc?tctgcgcatg?catagtcatc???120
ggacacctcc?tggaggagaa?tagatggatg?aacgagtcca?tcaccgctct?agccatcggg???180
ttgtgcactg?gcgttgtgat?tcttctgacg?acaaatggga?agagctctca?tatattcgta???240
ttcagcgaag?atctcttctt?catctacctc?ctccctccaa?tcatcttcaa?cgccgggttt???300
caagtaaaaa?agaaacaatt?tttccgcaac?ttcatgacaa?ttatgctgtt?tggtgcagtt???360
gggaccctca?tctcctttgt?cataatttct?cttggtgcaa?ttggtttatt?cagaaaaatg???420
aacataggtc?cactggaagt?tggagatttt?cttgcaattg?gggcaatctt?ttctgcaaca???480
gattctgttt?gcaccttgca?gattcttaac?caggatgaaa?caccattatt?atatagcttg???540
gtctttggtg?agggagttgt?caatgatgcc?acatcagtgg?tgcttttcaa?cgcaatacaa???600
aacttcgatc?ttgaacatat?tgatgcaggc?attgcagcaa?aatttattgg?aaatttcttt???660
tatttatttg?tcagcagcac?cttcttggga?gcatttgctg?gattgcttag?tgcgtatatc???720
ataaaaaagt?tgtattatgg?aaggcattct?actgatcgtg?aagttgcact?catgatgctt???780
atggcgtacc?tttcatatat?gctggctgag?ctgttagatc?taagtggtat?cctcacggtt???840
ttcttctgcg?gcatagtaat?gtcccattat?acgtggcaca?atgtgacaga?gagttcgaga???900
gtcacaacca?agcatgcttt?tgcaaccttg?tcatttattt?cggagatatt?cctcttcctt???960
tatgttggca?tggatgcatt?agacattgaa?aagtggagat?ttgtcagcga?cagtcctggg???1020
aaatcaattg?gtgttagcgc?aatcataatt?ggtttagttt?tgattggaag?agcagctttc???1080
gtcttcccgc?tgtctttcct?atccaactta?gctaagaagt?cccctaatga?aaaaatcacc???1140
ttcaaccagc?aagttaccat?atggtgggca?ggtttgatga?gaggtgctgt?atcaattgca???1200
cttgcttaca?atcagttcac?aagagctggt?catactcaac?ttccagggaa?tgcaatgatg???1260
atcaccagta?ccatcacagt?tgtgctattt?agcactgtgg?tattcggatt?attgacaaag???1320
cctttgataa?ggctcctgtt?gcctcctagt?acaaagcatc?ttaccggcag?ttgcagtttc???1380
aactctgagc?cctctacccc?aaaattttta?tccgctgcac?tactagaaaa?tattccagga???1440
tttgaagcag?aaggaggaac?aacaaacctt?ggggctcggc?cttcaagcc?ttcgaatgctc???1500
cttatgaagc?caactcacac?ggttcattac?tattggcgca?agtttgatga?tgctttcatg???1560
cgtccaatgt?ttggagggcg?aggctttgtt?ccttttgttc?ccggttcacc?tactgaacaa???1620
agtgttcatg?gatgggaatg?a?????????????????????????????????????????????1641
SEQ?ID?NO.2
＜110〉Shandong University
＜120〉sword-like iris vacuolar membrane Na
+/ H
+ Reverse transport protein 2 genes and application
<141>2005-5-13
<170>PatentIn?Version?2.1
<210>2
<211>546
<212>PRT
＜213〉sword-like iris vacuolar membrane Na
+/ H
+ Reverse transport protein 2 aminopeptidase gene acid sequences
<400>
MGSGMEDLLV?RLGVVSSTSD?HASVVSINLF?VALLCACIVI?GHLLEENRWM?NES?ITALAIG???60
LCTGVVILLT?TNGKSSHIFV?FSEDLFFIYL?LPPIIFNAGF?QVKKKQFFRN?FMTIMLFGAV????120
GTLISFVIIS?LGAIGLFRKM?NIGPLEVGDF?LAIGAIFSAT?DSVCTLQILN?QDETPLLYSL????180
VFGEGVVNDA?TSVVLFNAIQ?NFDLEHIDAG?IAAKFIGNFF?YLFVSSTFLG?AFAGLLSAYI????240
IKKLYYGRHS?TDREVALMML?MAYLSYMLAE?LLDLSGILTV?FFCGIVMSHY?TWHNVTESSR????300
VTTKHAFATL?SFISEIFLFL?YVGMDALDIE?KWRFVSDSPG?KSIGVSAIII?GLVLIGRAAF????360
VFPLSFLSNL?AKKSPNEKIT?FNQQVTIWWA?GLMRGAVSIA?LAYNQFTRAG?HTQLPGNAMM????420
ITSTITVVLF?STVVFGLLTK?PLIRLLLPPS?TKHLTGSCSF?NSEPSTPKFL?SAALLENIPG????480
FEAEGGTTNL?GARPSSLRML?LMKPTHTVHY?YWRKFDDAFM?RPMFGGRGFV?PFVPGSPTEQ????540
SVHGWE???????????????????????????????????????????????????????????????546
Claims (8)
1, sword-like iris vacuolar membrane Na
+/ H
+Reverse transport protein 2 genes is characterized in that having the sequence shown in SEQ ID NO.1:
ATGGGGTCTG?GTATGGAGGA?TCTGCTGGTG?AGGCTGGGTG?TCGTGTCGTC?CACCTCCGAC????60
CATGCCTCCG?TGGTCTCCAT?CAACCTCTTC?GTAGCTCTCC?TCTGCGCATG?CATAGTCATC???120
GGACACCTCC?TGGAGGAGAA?TAGATGGATG?AACGAGTCCA?TCACCGCTCT?AGCCATCGGG???180
TTGTGCACTG?GCGTTGTGAT?TCTTCTGACG?ACAAATGGGA?AGAGCTCTCA?TATATTCGTA???240
TTCAGCGAAG?ATCTCTTCTT?CATCTACCTC?CTCCCTCCAA?TCATCTTCAA?CGCCGGGTTT???300
CAAGTAAAAA?AGAAACAATT?TTTCCGCAAC?TTCATGACAA?TTATGCTGTT?TGGTGCAGTT???360
GGGACCCTCA?TCTCCTTTGT?CATAATTTCT?CTTGGTGCAA?TTGGTTTATT?CAGAAAAATG???420
AACATAGGTC?CACTGGAAGT?TGGAGATTTT?CTTGCAATTG?GGGCAATCTT?TTCTGCAACA???480
GATTCTGTTT?GCACCTTGCA?GATTCTTAAC?CAGGATGAAA?CACCATTATT?ATATAGCTTG???540
GTCTTTGGTG?AGGGAGTTGT?CAATGATGCC?ACATCAGTGG?TGCTTTTCAA?CGCAATACAA???600
AACTTCGATC?TTGAACATAT?TGATGCAGGC?ATTGCAGCAA?AATTTATTGG?AAATTTCTTT???660
TATTTATTTG?TCAGCAGCAC?CTTCTTGGGA?GCATTTGCTG?GATTGCTTAG?TGCGTATATC???720
ATAAAAAAGT?TGTATTATGG?AAGGCATTCT?ACTGATCGTG?AAGTTGCACT?CATGATGCTT???780
ATGGCGTACC?TTTCATATAT?GCTGGCTGAG?CTGTTAGATC?TAAGTGGTAT?CCTCACGGTT???840
TTCTTCTGCG?GCATAGTAAT?GTCCCATTAT?ACGTGGCACA?ATGTGACAGA?GAGTTCGAGA???900
GTCACAACCA?AGCATGCTTT?TGCAACCTTG?TCATTTATTT?CGGAGATATT?CCTCTTCCTT???960
TATGTTGGCA?TGGATGCATT?AGACATTGAA?AAGTGGAGAT?TTGTCAGCGA?CAGTCCTGGG??1020
AAATCAATTG?GTGTTAGCGC?AATCATAATT?GGTTTAGTTT?TGATTGGAAG?AGCAGCTTTC??1080
GTCTTCCCGC?TGTCTTTCCT?ATCCAACTTA?GCTAAGAAGT?CCCCTAATGA?AAAAATCACC??1140
TTCAACCAGC?AAGTTACCAT?ATGGTGGGCA?GGTTTGATGA?GAGGTGCTGT?ATCAATTGCA??1200
CTTGCTTACA?ATCAGTTCAC?AAGAGCTGGT?CATACTCAAC?TTCCAGGGAA?TGCAATGATG??1260
ATCACCAGTA?CCATCACAGT?TGTGCTATTT?AGCACTGTGG?TATTCGGATT?ATTGACAAAG??1320
CCTTTGATAA?GGCTCCTGTT?GCCTCCTAGT?ACAAAGCATC?TTACCGGCAG?TTGCAGTTTC??1380
AACTCTGAGC?CCTCTACCCC?AAAATTTTTA?TCCGCTGCAC?TACTAGAAAA?TATTCCAGGA??1440
TTTGAAGCAG?AAGGAGGAAC?AACAAACCTT?GGGGCTCGGC?CTTCAAGCCT?TCGAATGCTC??1500
CTTATGAAGC?CAACTCACAC?GGTTCATTAC?TATTGGCGCA?AGTTTGATGA?TGCTTTCATG??1560
CGTCCAATGT?TTGGAGGGCG?AGGCTTTGTT?CCTTTTGTTC?CCGGTTCACC?TACTGAACAA??1620
AGTGTTCATG?GATGGGAATG?A????????????????????????????????????????????1641
Wherein, comprise with sequence shown in the SEQ ID NO.1 having the sequence of at least 95% homogeny.
2, sword-like iris vacuolar membrane Na
+/ H
+Reverse transport protein 2 genes is characterized in that having aminoacid sequence shown in sequence table SEQ IDNO.2:
MGSGMEDLLV?RLGVVSSTSD?HASVVSINLF?VALLCACIVI?GHLLEENRWM?NESITALAIG????60
LCTGVVILLT?TNGKSSHIFV?FSEDLFFIYL?LPPIIFNAGF?QVKKKQFFRN?FMTIMLFGAV???120
GTLISFVIIS?LGAIGLFRKM?NIGPLEVGDF?LAIGAIFSAT?DSVCTLQILN?QDETPLLYSL???180
VFGEGVVNDA?TSVVLFNAIQ?NFDLEHIDAG?IAAKFIGNFF?YLFVSSTFLG?AFAGLLSAYI???240
IKKLYYGRHS?TDREVALMML?MAYLSYMLAE?LLDLSGILTV?FFCGIVMSHY?TWHNVTESSR???300
VTTKHAFATL?SFISEIFLFL?YVGMDALDIE?KWRFVSDSPG?KSIGVSAIII?GLVLIGRAAF???360
VFPLSFLSNL?AKKSPNEKIT?FNQQVTIWWA?GLMRGAVSIA?LAYNQFTRAG?HTQLPGNAMM???420
ITSTITVVLF?STVVFGLLTK?PLIRLLLPPS?TKHLTGSCSF?NSEPSTPKFL?SAALLENIPG???480
FEAEGGTTNL?GARPSSLRML?LMKPTHTVHY?YWRKFDDAFM?RPMFGGRGFV?PFVPGSPTEQ???540
SVHGWE?????????????????????????????????????????????????????546
Wherein, comprise with sequence shown in the SEQ ID NO.2 having the sequence of at least 95% homogeny.
3, sword-like iris vacuolar membrane Na according to claim 1
+/ H
+Reverse transport protein 2 genes is characterized in that, described sword-like iris vacuolar membrane Na
+/ H
+Reverse transport protein 2 genes can be building up in the prokaryotic expression carrier, by research method well known in the art with sword-like iris vacuolar membrane Na
+/ H
+Reverse transport protein 2 genes import prokaryotic organism, and by inducing, great expression goes out sword-like iris vacuolar membrane Na
+/ H
+Reverse transport protein 2.
4, according to claim 1 or 3 described sword-like iris vacuolar membrane Na
+/ H
+Reverse transport protein 2 genes is characterized in that, described sword-like iris vacuolar membrane Na
+/ H
+Reverse transport protein 2 can be used for studying sword-like iris vacuolar membrane Na
+/ H
+The structure of reverse transport protein 2, function and Antibody Preparation.
5, sword-like iris vacuolar membrane Na according to claim 1
+/ H
+Reverse transport protein 2 genes is characterized in that, described sword-like iris vacuolar membrane Na
+/ H
+Reverse transport protein 2 genes can pass through microinjection, particle gun, agriculture bacillus mediated, pollen tube channel method with sword-like iris vacuolar membrane Na
+/ H
+Reverse transport protein 2 genes change crop and herbage over to, cultivate the colory kind of anti-salt/be.
6, sword-like iris vacuolar membrane Na according to claim 1 or 5
+/ H
+Reverse transport protein 2 genes is characterized in that, described sword-like iris vacuolar membrane Na
+/ H
+Reverse transport protein 2 genes can be building up to any based in the Ti-plasmid binary vector, by research method well known in the art with sword-like iris vacuolar membrane Na
+/ H
+Reverse transport protein 2 genes import in the Agrobacterium, carry out Agrobacterium-mediated Transformation.
7, sword-like iris vacuolar membrane Na according to claim 6
+/ H
+Reverse transport protein 2 genes is characterized in that, with described sword-like iris vacuolar membrane Na
+/ H
+Reverse transport protein 2 genes are structured on the Ti-plasmid binary vector pBIUbiGFP of expression of plants as goal gene; The T-DNA25bp tumor-necrosis factor glycoproteins that LB and RB are wherein arranged, the promotor P-nos of rouge alkali synthetase gene and terminator T-nos also have the nptII that uses as selective marker in eukaryote, and the microbiotic that is used to select is kantlex and G418; In pBIUbiGFP, also have corn ubiquitin super promoter Ubi and green fluorescent protein GFP gene; Sword-like iris vacuolar membrane Na
+/ H
+Reverse transport protein 2 genes are inserted on the Ubiquitin promotor and the multiple clone site between the GFP gene of pBIUbiGFP plasmid, and form fusion gene with the GFP gene, express a N-terminal and are connected with the proteic sword-like iris vacuolar membrane Na of GFP
+/ H
+Reverse transport protein 2.
8, sword-like iris vacuolar membrane Na according to claim 7
+/ H
+Reverse transport protein 2 genes is characterized in that, described double base transforms recombinant vectors pBIUbiGFP:I1NHX2 and can transform in Agrobacterium AGL1, infects wheat by Agrobacterium shoot apex conversion method, thereby obtain transgenic wheat.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200510043511 CN1706951A (en) | 2005-05-13 | 2005-05-13 | Tonoplast Membrane Na+/H+ Antiporter 2 Gene and Its Application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200510043511 CN1706951A (en) | 2005-05-13 | 2005-05-13 | Tonoplast Membrane Na+/H+ Antiporter 2 Gene and Its Application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1706951A true CN1706951A (en) | 2005-12-14 |
Family
ID=35581061
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 200510043511 Pending CN1706951A (en) | 2005-05-13 | 2005-05-13 | Tonoplast Membrane Na+/H+ Antiporter 2 Gene and Its Application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1706951A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105189534A (en) * | 2013-06-24 | 2015-12-23 | 创世纪种业有限公司 | A sodium-hydrogen antiporter protein NHX3 and its coding gene and application |
-
2005
- 2005-05-13 CN CN 200510043511 patent/CN1706951A/en active Pending
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105189534A (en) * | 2013-06-24 | 2015-12-23 | 创世纪种业有限公司 | A sodium-hydrogen antiporter protein NHX3 and its coding gene and application |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101063138A (en) | Plant flower organ specificity promoter and its application | |
| CN1706951A (en) | Tonoplast Membrane Na+/H+ Antiporter 2 Gene and Its Application | |
| CN1699568A (en) | Tall Fescue Tonoplast Na+/H+ Antiporter 1 Gene and Its Application | |
| CN1291021C (en) | Use of boea crassifolia BcBCP1 gene for breeding drought-salt-tolerant plants | |
| CN1188523C (en) | Blue-green alga shuttle plasmid expression vector and method for expressing thymison 'alpha' 1 | |
| CN1818065A (en) | Cotton GhZFP1 gene sequence, its clone and use | |
| CN1218959C (en) | Paddy rice ethylene receptor protein, coded gene and use thereof | |
| CN1324137C (en) | Salt resistant and drought resistant gene of Thellugielkla halophila and its coded protein and use | |
| CN1824779A (en) | A kind of soybean heat shock transcription factor and its coding gene and application | |
| CN100351380C (en) | APDHI sequence of DNA unwindase gene of kender and its clone and applciation thereof | |
| CN1296383C (en) | Plant DREB transcription factor and its coding gene and uses | |
| CN1239707C (en) | Anti longicorn Bt gene and its application | |
| CN1844397A (en) | Phophomannose isomerase gene and its coded protein and use | |
| CN1184231C (en) | Wheat TaDREB, its code gene and method for culturing adverse-resistant plant | |
| CN1632122A (en) | Alfalfa Na+/H+ Antiporter Gene and Its Cloning and Application | |
| CN1769457A (en) | Phalaenopsis pPI15 coding sequence and its application | |
| CN1908011A (en) | Plant inverse-resistant zinc finger protein, coding gene and application thereof | |
| CN1614024A (en) | Transgened-tobacco for degrading organic mercury pollution | |
| CN1258595C (en) | Phloem-specific expression promoter | |
| CN1699569A (en) | Tonoplast Membrane Na+/H+ Antiporter 2 Gene and Its Application in Wheatgrass | |
| CN1888071A (en) | Chuancao-II Laomangmai wheat pest-resisting gene genetically modifying technology | |
| CN1844143A (en) | A plant salt-tolerance-related protein, its coding gene and application | |
| CN1768577A (en) | A method of cultivating fusarium wilt-resistant bananas | |
| CN1238374C (en) | Na+/H+ retrograde transport protein of New Zedland spinach, its coding gene and application | |
| CN1765924A (en) | Plant Stress Resistance Related Proteins and Their Encoding Genes and Applications |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |