CN103130882B - Betaine transporter, encoding gene and applications thereof - Google Patents
Betaine transporter, encoding gene and applications thereof Download PDFInfo
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- CN103130882B CN103130882B CN201110379442.8A CN201110379442A CN103130882B CN 103130882 B CN103130882 B CN 103130882B CN 201110379442 A CN201110379442 A CN 201110379442A CN 103130882 B CN103130882 B CN 103130882B
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Abstract
The present invention relates to a betaine transporter, which has an amino acid sequence 1 represented by SEQ ID No:2, or has the following amino acid sequence 2, wherein an amino acid sequence 1 represented by SEQ ID No:2 is subjected to substituting, deleting, or adding of one or a plurality of amino acid residues to form the amino acid sequence 2, and the amino acid sequence 2 has betaine transporter activity. In addition, the invention further relates to an encoding gene of the betaine transporter, wherein the gene has a nucleotide sequence represented by SEQ ID No:1, or a nucleotide sequence encoding the amino acid sequence 1 represented by SEQ ID No:2. The invention further relates to a recombinant vector containing the betaine transporter encoding gene and cells containing the betaine transporter encoding gene, and applications of the betaine transporter, the encoding gene, the recombinant vector containing the gene and the cells containing the gene in alkali tolerance transgene organism cultures.
Description
Technical field
The present invention relates to a kind of trimethyl-glycine translocator, the encoding gene that also relates to trimethyl-glycine translocator, and the recombinant vectors that contains trimethyl-glycine transporter gene and cell, and recombinant vectors and cell that trimethyl-glycine translocator and encoding gene thereof contain this gene have the application in alkali resistance genetically modified organism in cultivation.
Background technology
Trimethyl-glycine translocator (betaine transporter) is in cell, to be responsible for a kind of cross-film transport protein of transhipment trimethyl-glycine, is extensively present in bacterium and higher organism.Current research shows, trimethyl-glycine has several functions, as methyl donor, has promotion Animal lipid metabolism, mitigation stress, regulate osmotic pressure, improve a poor appetite, stable vitamin, prevention coccidiosis, the effects such as efficiency of feed utilization that improve.
As the trimethyl-glycine of one of compatible solute main in biomass cells, maintaining Premeabilisation of cells pressure, the persistence of the protection of biological enzyme and anti-salt is had to important effect.When being subject to saline and alkaline or water stress, in tenuigenin, accumulate a large amount of Organic osmoticas as trimethyl-glycine, and be mainly to help to press to vacuole by the inorganic osmotica in tenuigenin, making kytoplasm and intracellular fluid bubble) external environment maintains osmotic equilibrium, avoided like this murder by poisoning of tenuigenin high density inorganic ion-pair enzyme and metabolism.
Summary of the invention
The molecular biology research of the present inventor based on to trimethyl-glycine translocator, be surprised to find that from Alkaliphilic bacillus (N16-5, CGMCC NO.0369) a kind of trimethyl-glycine translocator and encoding gene thereof in, have been obtained, also provide on the other hand the recombinant vectors and the cell that contain trimethyl-glycine transporter gene, and recombinant vectors and cell that trimethyl-glycine translocator and encoding gene thereof contain this gene has the application in alkali resistance genetically modified organism in cultivation.
The invention provides a kind of trimethyl-glycine translocator, wherein, this trimethyl-glycine translocator has the aminoacid sequence shown in SEQ ID No:2, or this trimethyl-glycine translocator 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 trimethyl-glycine translocator activity.
The present invention also provides a kind of trimethyl-glycine transporter 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 trimethyl-glycine transporter gene provided by the invention.
The present invention also provides a kind of transgenic cell, and wherein, this transgenic cell contains trimethyl-glycine transporter gene provided by the invention.
The present invention also provides the trimethyl-glycine translocator 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.
Trimethyl-glycine translocator has important application potential at aspects such as saline alkali tolerant plant cultivations.By clone, obtain trimethyl-glycine transporter gene, and by genetically modified operation, microbe-derived trimethyl-glycine translocator 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-Bspn165-opuD) that recombinant vectors pUC18-Bspn165-opuD 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-Bspn165-opuD) that recombinant vectors pUC18-Bspn165-opuD is imported is inoculated into respectively pH, 5N NaOH adjusting), in LB liquid nutrient medium (100 μ g/ml penbritin), cultivate and measure OD after 12 hours
600, the e. coli k12 that imports pUC18 empty carrier of take is contrast (three every group parallel).
Fig. 2 has shown (pUC18-Bspn165-opuD) alkali-proof measurement result of the e. coli k12 (Δ opuD) of trimethyl-glycine translocator disappearance and the K12 (Δ opuD) of importing recombinant vectors pUC18-Bspn165-opuD, wherein, by the K12 (Δ opuD) that imports recombinant vectors pUC18-Bspn165-opuD (pUC18-Bspn165-opuD) and the e. coli k12 (Δ opuD) of trimethyl-glycine translocator disappearance to be inoculated into respectively pH be 8.0, 8.5, 9.0 and the 9.5 (CAPS that contain 50mM, HEPES and TRICINE, 5N NaOH adjusting) in LB liquid nutrient medium, cultivate and measure OD after 12 hours
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 trimethyl-glycine translocator, wherein, this trimethyl-glycine translocator has the aminoacid sequence shown in SEQID No:2, or this trimethyl-glycine translocator 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 trimethyl-glycine translocator activity.Preferably, described trimethyl-glycine translocator has the aminoacid sequence shown in SEQ ID No:2.
Correspondingly, the present invention also provides a kind of trimethyl-glycine transporter 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.Preferably, described gene has the nucleotide sequence shown in SEQ ID No:1.
Trimethyl-glycine transporter 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 trimethyl-glycine transporter 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 trimethyl-glycine transporter 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 trimethyl-glycine translocator provided by the invention and encoding gene thereof and the recombinant vectors that contains trimethyl-glycine transporter 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 trimethyl-glycine translocator
(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 trimethyl-glycine transporter gene
Analyze Alkaliphilic bacillus (N16-5, CGMCC NO.0369) after genomic information, the upstream and downstream primer of design trimethyl-glycine transporter gene, wherein upstream primer is 5 '-TATGACCATGATTACCATGAAAAAAGTGTCC-3 ', and downstream primer is 5 '-CAGGTCGACTCTAGATTAAGTCGGTTTAATT-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 trimethyl-glycine transporter 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 1551bp.
The gene function checking of embodiment 2 coding trimethyl-glycine translocators
(1) structure of recombinant cloning vector pUC18-Bspn165-opuD
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-Bspn165-opuD 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-Bspn165-opuD) that recombinant vectors pUC18-Bspn165-opuD inserts, and confirm that the nucleotide sequence of extension increasing sequence that pUC18-Bspn165-opuD inserts and trimethyl-glycine translocator is in full accord through order-checking.
(2) structure of e. coli k12 BW25113 trimethyl-glycine translocator 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 trimethyl-glycine transporter gene of e. coli k12 BW2511, obtain the mutant E.coli K12 (Δ opuD) that e. coli k12 BW25113 trimethyl-glycine transporter gene (Δ opuD) lacks completely, trimethyl-glycine transporter gene is replaced by kanamycin gene.
(3) the alkali-proof mensuration of the e. coli k12 (pUC18-Bspn165-opuD) that recombinant vectors pUC18-Bspn165-opuD 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-Bspn165-opuD) that recombinant vectors pUC18-Bspn165-opuD is imported is inoculated into respectively pH, 5N NaOH adjusting), in LB liquid nutrient medium (100 μ g/ml penbritin), cultivate and measure OD after 12 hours
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) K12 (Δ opuD) of the e. coli k12 (Δ opuD) that trimethyl-glycine translocator lacks and importing recombinant vectors pUC18-Bspn165-opuD is alkali-proof mensuration (pUC18-Bspn165-opuD)
By the K12 (Δ opuD) that imports recombinant vectors pUC18-Bspn165-opuD (pUC18-Bspn165-opuD) and the e. coli k12 (Δ opuD) of trimethyl-glycine translocator disappearance to be inoculated into respectively pH be 8.0,8.5,9.0 and 9.5 (CAPS that contains 50mM, HEPES and TRICINE, 5N NaOH adjusting), in LB liquid nutrient medium, cultivate and measure OD after 12 hours
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-Bspn165-opuD 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 trimethyl-glycine transporter gene (Bspn165-opuD) can improve the alkali resistance of e. coli k12.
As can be seen from Figure 2 the e. coli k12 (Δ opuD) that, imports recombinant vectors pUC18-Bspn165-opuD is at pH 8.0,8.5,9.0 and 9.5 (contain 50mM CAPS, HEPES and TRICINE, 5N NaOH regulates) LB liquid nutrient medium in, after 12 hours, measure OD
600apparently higher than the e. coli k12 that imports pUC18 empty carrier, this has also proved that the importing of trimethyl-glycine transporter gene (Bspn165-opuD) can improve the alkali resistance of e. coli k12 (Δ opuD).
In sum, the trimethyl-glycine transporter gene that the application provides has important application potential at the aspects such as cultivation of Salt And Alkali Tolerance biology.By clone, obtain trimethyl-glycine transporter gene, and by genetically modified operation, microbe-derived trimethyl-glycine translocator is imported in vegetable cell, the transfer-gen plant that obtains the raising of salt tolerant alkalescence becomes possibility.
Claims (7)
1. a trimethyl-glycine translocator, is characterized in that, the aminoacid sequence of this trimethyl-glycine translocator is as shown in SEQ ID No:2.
2. a trimethyl-glycine transporter gene, is characterized in that, 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.
6. transgenic cell according to claim 5, wherein, described transgenic cell is prokaryotic cell prokaryocyte or eukaryotic cell.
7. trimethyl-glycine translocator claimed in claim 1, gene claimed in claim 2, recombinant vectors claimed in claim 4 have the application in alkali resistance genetically modified organism in cultivation;
Wherein, described biology is intestinal bacteria.
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Non-Patent Citations (6)
| Title |
|---|
| Cloning and characterizaiton of the halobacillus trueperi betH gene,encoding the transport system for the compatible solute glycine betaine;Lu Weidong等;《Fems microbiology letters》;20041231;第235卷;393-399 * |
| LuWeidong等.CloningandcharacterizaitonofthehalobacillustrueperibetHgene encoding the transport system for the compatible solute glycine betaine.《Fems microbiology letters》.2004 |
| Saier MH jr等.Transport capabilities encoded within the bacillus subtilis genome.《J mol microbiol biotechnol》.2002,第4卷(第1期),37-67. |
| Transport capabilities encoded within the bacillus subtilis genome;Saier MH jr等;《J mol microbiol biotechnol》;20020604;第4卷(第1期);37-67 * |
| 嗜碱Bacillus sp.N16-5不同碳源条件下比较蛋白质组分析;李刚 等;《中国科学》;20101231;第40卷(第12期);1117-1127 * |
| 李刚 等.嗜碱Bacillus sp.N16-5不同碳源条件下比较蛋白质组分析.《中国科学》.2010,第40卷(第12期),1117-1127. |
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