CN103087162B - Sodium hydrogen pump protein, its coding gene, and applications thereof - Google Patents
Sodium hydrogen pump protein, its coding gene, and applications thereof Download PDFInfo
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- CN103087162B CN103087162B CN201110337942.5A CN201110337942A CN103087162B CN 103087162 B CN103087162 B CN 103087162B CN 201110337942 A CN201110337942 A CN 201110337942A CN 103087162 B CN103087162 B CN 103087162B
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Abstract
The invention relates to an Na<+>/H<+>antiporter protein. The Na<+>/H<+>antiporter protein has an amino acid sequence represented by SEQ ID No:2, or has an amino acid sequence obtained through substituting, deleting or adding one or more amino acid residues to the amino acid sequence represented by the SEQ ID No:2 and still having Na<+>/H<+>antiporter protein activities. The invention also relates to a coding gene of the Na<+>/H<+>antiporter protein. The gene has a nucleotide sequence represented by SEQ ID No:1, or has a nucleotide sequence coding the amino acid sequence represented by SEQ ID No:2. The invention further relates to a recombinant vector containing the Na<+>/H<+>antiporter gene, a cell containing the Na<+>/H<+>antiporter gene, and applications of the Na<+>/H<+>antiporter protein, the coding gene thereof, the recombinant vector containing the gene and the cell containing the gene in the cultivation of alkali-resistant transgenic organisms.
Description
Technical field
The present invention relates to a kind of sodium hydrogen pump albumen, the encoding gene that also relates to sodium hydrogen pump albumen, and the recombinant vectors and the cell that contain sodium hydrogen pump gene, and recombinant vectors and cell that sodium hydrogen pump albumen and encoding gene thereof contain this gene have the application in alkali resistance genetically modified organism in cultivation.
Background technology
Sodium hydrogen pump (Na
+/ H
+antiporter) be in cell, to be responsible for Na
+/ H
+a kind of cross-film transport protein of exchange, is extensively present in the plasma membrane of bacterium, people and higher plant and the film of many eukaryotic cells devices.Plasma membrane H
+-ATPase uses the energy of hydrolysising ATP H
+from tenuigenin, pump cell, produce the H across plasma membrane
+gradient of electrochemical potential, provides energy, drives the sodium hydrogen pump albumen on plasma membrane, makes H
+along its electrochemical potential, enter cell, simultaneously Na
+contrary its electrochemical potential is discharged cell.Sodium hydrogen pump albumen passes through Na
+outer row keeps intracellular low Na
+stablizing of level and pH value, is that biomass cells tolerates saline and alkaline key factor, and it,, in the generation and ion balancing procedure of organoid, also carries out the function of volume and osmoregulation in addition, is the key factor that keeps cell plasma equilibrium.
Summary of the invention
The molecular biology research of the present inventor based on to sodium hydrogen pump, discovery is from Alkaliphilic bacillus (N16-5, CGMCC NO.0369) a kind of sodium hydrogen pump albumen and encoding gene thereof in, have been obtained, also provide on the other hand the recombinant vectors and the cell that contain sodium hydrogen pump gene, and recombinant vectors and cell that sodium hydrogen pump albumen and encoding gene thereof contain this gene has the application in alkali resistance genetically modified organism in cultivation.
The invention provides a kind of sodium hydrogen pump albumen, wherein, this sodium hydrogen pump albumen has the aminoacid sequence shown in SEQ ID No:2, or this sodium hydrogen pump albumen has the aminoacid sequence shown in SEQ ID No:2 through the replacement of one or several amino-acid residue, disappearance or after adding, still has the aminoacid sequence of sodium hydrogen pump protein-active.
The present invention also provides a kind of sodium hydrogen pump gene, and wherein, this gene has the nucleotide sequence shown in SEQ ID No:1, or this gene has the nucleotide sequence of the aminoacid sequence shown in coding SEQ ID No:2.
In addition, the present invention also provides a kind of recombinant vectors, and wherein, this recombinant vectors contains sodium hydrogen pump gene provided by the invention.
The present invention also provides a kind of transgenic cell, and wherein, this transgenic cell contains sodium hydrogen pump gene provided by the invention.
The present invention also provides the sodium hydrogen pump albumen and encoding gene, the recombinant vectors that contains this gene and the transgenic cell that obtain to have the application in alkali resistance genetically modified organism in cultivation.
Sodium hydrogen pump albumen has important application potential at aspects such as saline alkali tolerant plant cultivations.By clone, obtain sodium hydrogen pump gene, and by genetically modified operation, microbe-derived sodium hydrogen pump gene is imported in vegetable cell, the transfer-gen plant that obtains the raising of salt tolerant alkalescence becomes possibility.
Accompanying drawing explanation
Fig. 1 has shown the alkali-proof measurement result of the e. coli k12 (pUC18-Bsp165-NhaE) that recombinant vectors pUC18-Bsp165-NhaE imports, wherein, it is 8.0,8.5,9.0 and 9.5 (CAPS that contains 50mM, HEPES and TRICINE that the e. coli k12 (pUC18-Bsp165-NhaE) that recombinant vectors pUC18-Bsp165-NhaE is imported is inoculated into respectively pH, 5N NaOH adjusting), in LB liquid nutrient medium (100 μ g/ml penbritin), cultivate after 12 hours and measure OD
600, the e. coli k12 that imports pUC18 empty carrier of take is contrast (three every group parallel).
Fig. 2 has shown the intestinal bacteria KNabc of sodium hydrogen pump disappearance and the alkali-proof measurement result of KNabc (pUC18-Bsp165-NhaE) that imports recombinant vectors pUC18-Bsp165-NhaE, wherein, it is 8.0,8.5,9.0 and 9.5 (CAPS that contains 50mM, HEPES and TRICINE that the intestinal bacteria KNabc of the KNabc (pUC18-Bsp165-NhaE) of importing recombinant vectors pUC18-Bsp165-NhaE and sodium hydrogen pump disappearance is inoculated into respectively to pH, 5N NaOH adjusting), in LB liquid nutrient medium, cultivate after 12 hours and measure OD
600, take e. coli k12 as contrast (three every group parallel).
Embodiment
Following the specific embodiment of the present invention is elaborated.Should be understood that, embodiment described herein only, for description and interpretation the present invention, is not limited to the present invention.
The invention provides a kind of sodium hydrogen pump albumen, wherein, this sodium hydrogen pump albumen has the aminoacid sequence shown in SEQ ID No:2, or this sodium hydrogen pump albumen has the aminoacid sequence shown in SEQ ID No:2 through the replacement of one or several amino-acid residue, disappearance or after adding, still has the aminoacid sequence of sodium hydrogen pump protein-active.Preferably, described sodium hydrogen pump albumen has the aminoacid sequence shown in SEQ ID No:2.
Correspondingly, the present invention also provides a kind of sodium hydrogen pump gene, and wherein, this gene has the nucleotide sequence shown in SEQ IDNo:1, or this gene has the nucleotide sequence of the aminoacid sequence shown in coding SEQ ID No:2.Preferably, described gene has the nucleotide sequence shown in SEQ ID No:1.
Sodium hydrogen pump gene provided by the invention is that clone obtains from Alkaliphilic bacillus (N16-5, CGMCC NO.0369).
In addition, the present invention also provides a kind of recombinant vectors, and wherein, this recombinant vectors contains sodium hydrogen pump gene provided by the invention.
In the present invention, described " carrier " can select various carrier known in the art, as commercially available various plasmids, clay, phage and retrovirus etc.The preferred intestinal bacteria pUC18 of the present invention plasmid.
The present invention also provides a kind of transgenic cell, and wherein, this transgenic cell contains sodium hydrogen pump gene provided by the invention.Described transgenic cell is prokaryotic cell prokaryocyte or eukaryotic cell, can be preferably intestinal bacteria, Bacillus subtilus or tobacco BY2 cell, most preferably intestinal bacteria.
In addition, the present invention also provides sodium hydrogen pump albumen provided by the invention and encoding gene thereof and the recombinant vectors that contains sodium hydrogen pump gene and transgenic cell to have the application in alkali resistance genetically modified organism in cultivation.Described biology is plant or microorganism.
Embodiment 1
The clone of the nucleotide sequence of coding sodium hydrogen pump
(1) extraction and the purifying of the total DNA of Alkaliphilic bacillus (N16-5, CGMCC NO.0369)
Get Alkaliphilic bacillus (N16-5, CGMCC NO.0369) 20 grams of fresh wet thallus, be suspended from 10 milliliters of 50 mM/ls of Tris damping fluids (pH 8.0), add a small amount of N,O-Diacetylmuramidase and 8 milliliters 0.25 mM/l ethylenediamine tetraacetic acid (EDTA) (EDTA) (pH 8.0), after mixing, in 37 ℃, place 20 minutes, then add 2 milliliter of 10% sodium lauryl sulphate (SDS), place 5 minutes for 55 ℃, use respectively equal-volume phenol, each extracting of chloroform once, get the supernatant solution of last extracting, add 2 times of volume ethanol, precipitation DNA.The DNA that precipitation is reclaimed successively uses after 70 volume % ethanolic solns and absolute ethanol washing, gained DNA is dissolved in to 0.5 milliliter of TE damping fluid, and (pH 8.0,10 mM/ls of Tris, 1 mM/l of EDTA), add 10 mg/ml RNA enzyme (RNase) 3 microlitres, 37 ℃ are incubated 1 hour, use respectively equal-volume phenol, each extracting of chloroform once, get supernatant liquor and add 2 times of volume ethanol, precipitation reclaims DNA, successively uses after 70 volume % ethanolic solns and absolute ethanol washing vacuum-drying DNA precipitation, with deionized water dissolving, obtain total DNA solution.The ultraviolet spectrophotometer measurement result of DNA solution is A
260/ A
280=1.818, A
260/ A
230=2.052.
(2) clone of sodium hydrogen pump gene
Analyze Alkaliphilic bacillus (N16-5, CGMCC NO.0369) after genomic information, the upstream and downstream primer of design sodium hydrogen pump gene, wherein upstream primer is 5 '-TATGACCATGATTACCATGACATACGCTC-3 ', and downstream primer is 5 '-CAGGTCGACTCTAGATTATTTTTTTGCTTTT-3 '.By the nucleic acid polymerase Pyrobest (Takara) of high-fidelity, with Alkaliphilic bacillus (N16-5, the CGMCC NO.0369) genome of said extracted, be the total length that template amplification goes out sodium hydrogen pump gene.By the size of 1% agarose gel electrophoresis testing goal gene, and PCR product is delivered to the order-checking of Nuo Sai genome company, finally obtain the nucleotide sequence as shown in SED ID NO:1 of 729bp.
The gene function checking of embodiment 2 coding sodium hydrogen pumps
(1) structure of recombinant cloning vector pUC18-pUC18-Bsp165-NhaE
Utilize Cycle-pure Kit purifying PCR product obtained above.Utilize Fast clone Kit that the PCR product of purifying is connected with pUC18, the recombinant vectors pUC18-Bsp165-NhaE building is imported in e. coli k12 BW25113 by chemical transformation.The screening of the LB flat board by containing 100 μ g/ml penbritins and PCR checking obtain containing the positive colony e. coli k12 (pUC18-Bsp165-NhaE) that recombinant vectors pUC18-Bsp165-NhaE inserts, and confirm that the nucleotide sequence of extension increasing sequence that pUC18-Bsp165-NhaE inserts and sodium hydrogen pump is in full accord through order-checking.
(2) structure of e. coli k12 BW25113 sodium hydrogen pump deletant
The primer amplification target practice gene that contains the DNA fragmentation of the upstream and downstream homology arm that needs to be knocked out gene by design, utilizes intestinal bacteria to enter phage and has the λ Red recombination system different with host goal gene is knocked out fast and accurately.By knocking out the sodium hydrogen pump gene of e. coli k12 BW2511, obtain the mutant E.coli KNabc that e. coli k12 BW25113 sodium hydrogen pump gene lacks completely, sodium hydrogen pump gene is replaced by kanamycin gene.
(3) the alkali-proof mensuration of the e. coli k12 (pUC18-Bsp165-NhaE) that recombinant vectors pUC18-Bsp165-NhaE imports
It is 8.0,8.5,9.0 and 9.5 (CAPS that contains 50mM, HEPES and TRICINE that the e. coli k12 (pUC18-Bsp165-NhaE) that recombinant vectors pUC18-Bsp165-NhaE is imported is inoculated into respectively pH, 5N NaOH adjusting), in LB liquid nutrient medium (100g/ml penbritin), cultivate after 12 hours and measure OD
600, the e. coli k12 that imports pUC18 empty carrier of take is contrast (three every group are parallel), result is as shown in Figure 1.
(4) the alkali-proof mensuration of KNabc (pUC18-Bsp165-NhaE) of the intestinal bacteria KNabc of sodium hydrogen pump disappearance and importing recombinant vectors pUC18-Bsp165-NhaE
It is 8.0,8.5,9.0 and 9.5 (CAPS that contains 50mM, HEPES and TRICINE that the intestinal bacteria KNabc of the KNabc (pUC18-Bsp165-NhaE) of importing recombinant vectors pUC18-Bsp165-NhaE and sodium hydrogen pump disappearance is inoculated into respectively to pH, 5N NaOH adjusting), in LB liquid nutrient medium, cultivate after 12 hours and measure OD
600, take e. coli k12 as contrast (three every group are parallel), result is as shown in Figure 2.
As can be seen from Figure 1, the e. coli k12 that imports recombinant vectors pUC18-Bsp165-NhaE is in 8.0,8.5,9.0 and 9.5 LB liquid nutrient medium (100 μ g/ml penbritin) at pH, after 12 hours, measures OD
600apparently higher than the e. coli k12 that imports pUC18 empty carrier, this has also proved that the importing of sodium hydrogen pump gene (Bsp165-NhaE) can improve the alkali resistance of e. coli k12.
As can be seen from Figure 2, import the intestinal bacteria KNabc of recombinant vectors pUC18-Bsp165-NhaE at pH 8.0,8.5, in the LB liquid nutrient medium of 9.0 and 9.5 (containing 50mM CAPS, HEPES and TRICINE, 5N NaOH adjusting), after 12 hours, measure OD
600apparently higher than the deletant KNabc that imports intestinal bacteria sodium hydrogen pump, this has also proved that the importing of sodium hydrogen pump gene (Bsp165-NhaE) can improve the alkali resistance of intestinal bacteria KNabc.
In sum, the sodium hydrogen pump gene that the application provides has important application potential at the aspects such as cultivation of Salt And Alkali Tolerance biology.By clone, obtain sodium hydrogen pump gene, and by genetically modified operation, microbe-derived sodium hydrogen pump is imported in vegetable cell, the transfer-gen plant that obtains the raising of salt tolerant alkalescence becomes possibility.
Claims (6)
1. a sodium hydrogen pump albumen, is characterized in that, the aminoacid sequence of this sodium hydrogen pump albumen is as shown in SEQ ID No:2.
2. a sodium hydrogen pump gene, is characterized in that, the nucleotide sequence of this gene is as shown in SEQ ID No:1, or the nucleotides sequence of this gene is classified the nucleotide sequence of the aminoacid sequence shown in coding SEQ ID No:2 as.
3. gene according to claim 2, wherein, the nucleotide sequence of this gene is as shown in SEQ ID No:1.
4. a recombinant vectors, is characterized in that, this recombinant vectors contains gene claimed in claim 2.
5. a transgenic cell, is characterized in that, this transgenic cell contains gene claimed in claim 2; Wherein, described transgenic cell is prokaryotic cell prokaryocyte.
6. sodium hydrogen pump albumen claimed in claim 1, gene claimed in claim 2, recombinant vectors claimed in claim 4, transgenic cell claimed in claim 5 have the application in alkali resistance genetically modified organism in cultivation; Wherein, described biology is prokaryotic organism.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1958796A (en) * | 2005-10-31 | 2007-05-09 | 中国科学院微生物研究所 | Nucleotide sequence protein gene of coded natrium hydrogen pump, and application |
| WO2007069782A1 (en) * | 2005-12-16 | 2007-06-21 | Ajinomoto Co., Inc. | L-amino acid producing bacterium and method of producing l-amino acid |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1958796A (en) * | 2005-10-31 | 2007-05-09 | 中国科学院微生物研究所 | Nucleotide sequence protein gene of coded natrium hydrogen pump, and application |
| WO2007069782A1 (en) * | 2005-12-16 | 2007-06-21 | Ajinomoto Co., Inc. | L-amino acid producing bacterium and method of producing l-amino acid |
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