CN103087160B - Flagellum motor protein, coding gene thereof, and applications thereof - Google Patents
Flagellum motor protein, coding gene thereof, and applications thereof Download PDFInfo
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- CN103087160B CN103087160B CN201110338601.XA CN201110338601A CN103087160B CN 103087160 B CN103087160 B CN 103087160B CN 201110338601 A CN201110338601 A CN 201110338601A CN 103087160 B CN103087160 B CN 103087160B
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Abstract
The invention relates to a flagellum motor protein. The flagellum motor protein has an amino acid sequence represented by SEQ ID No.2, or the flagellum motor protein has an amino acid sequence with flagellum motor protein activity, wherein the amino acid sequence is obtained by that the amino acid sequence represented by the SEQ ID No.2 is subjected to substitution, deletion, or addition of one or more amino acid residues. The invention also relates to a coding gene of the flagellum motor protein. The gene has a nucleotide sequence represented by SEQ ID No.1, or a nucleotide sequence coding the amino acid sequence represented by SEQ ID No.2. The invention also relates to a recombinant vector and a cell comprising the flagellum motor protein gene, and the applications of the flagellum motor protein, the coding gene thereof, and the recombinant vector and cell comprising the gene in culturing organisms with alkali resistance transgene.
Description
Technical field
The present invention relates to a kind of flagellar movement albumen, the encoding gene that also relates to flagellar movement albumen, and the recombinant vectors that contains flagellar movement protein gene and cell, and recombinant vectors and cell that flagellar movement albumen and encoding gene thereof contain this gene have the application in alkali resistance genetically modified organism in cultivation.
Background technology
On some bacterium thalline, there is elongated and crooked filament, be called flagellum (flagellum).If the length of flagellum often surpasses thousand times of thalline.On some thalline, with elongated and be the filament of wavy bending, 1-2 root, can reach hundreds of at most at least, is the locomotive organ of bacterium.Most bacteriums rely on flagellum to move, and the motion of flagellum is mainly to complete whole process by flagellar movement albumen (flagellar motor protein).
US 2008095793 has reported the application of chlamydozoan flagellin antigen aspect chlamydial control and diagnosis, the leading indicator of control using chlamydozoan flagellin as chlamydozoan.WO 2005089429 and US 2007218048 have reported that flagellum energy that the flagellum energy of sperm carries albumen and sperm carries the application of early motility of sperm diagnosis of albumen.
Research about flagellum and flagellin at present mainly concentrates on division bacteria evaluation, the aspects such as antibody of molecular diagnosis and flagellum epi-position.
Summary of the invention
The molecular biology research of the present inventor based on to flagellar movement albumen, be surprised to find that from basophilic Zymomonas mobilis (Alkalimonas amylolytica N10, CGMCC NO.0463) a kind of flagellar movement albumen and encoding gene thereof in, have been obtained, also provide on the other hand the recombinant vectors and the cell that contain flagellar movement protein gene, and recombinant vectors and cell that flagellar movement 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 flagellar movement albumen, wherein, this flagellar movement albumen has the aminoacid sequence shown in SEQ ID No:2, or this flagellar movement 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 flagellar movement protein-active.
The present invention also provides a kind of flagellar movement protein 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 flagellar movement protein gene provided by the invention.
The present invention also provides a kind of transgenic cell, and wherein, this transgenic cell contains flagellar movement protein gene provided by the invention.
The present invention also provides the flagellar movement 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.
Flagellar movement albumen has important application potential at aspects such as saline alkali tolerant plant cultivations.By clone, obtain flagellar movement protein gene, and by genetically modified operation, microbe-derived flagellar movement albumen 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-Aan10-motY) that recombinant vectors pUC18-Aan10-motY 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-Aan10-motY) that recombinant vectors pUC18-Aan10-motY 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 (pUC18-Aan10-motY) alkali-proof measurement result of the e. coli k12 (Δ mot) of flagellar movement protein delation and the K12 (Δ mot) of importing recombinant vectors pUC18-Aan10-motY, wherein, by the K12 (Δ Aan10-motY) that imports recombinant vectors pUC18-Aan10-motY (pUC18-Aan10-motY) and the e. coli k12 (Δ mot) of flagellar movement protein delation 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 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 flagellar movement albumen, wherein, this flagellar movement albumen has the aminoacid sequence shown in SEQ ID No:2, or this flagellar movement 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 flagellar movement protein-active.Preferably, described flagellar movement albumen has the aminoacid sequence shown in SEQ ID No:2.
Correspondingly, the present invention also provides a kind of flagellar movement protein 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.
Flagellar movement protein gene provided by the invention is that clone obtains from basophilic Zymomonas mobilis (Alkalimonas amylolytica N10, CGMCC NO.0463).
In addition, the present invention also provides a kind of recombinant vectors, and wherein, this recombinant vectors contains flagellar movement protein 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 flagellar movement protein 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 flagellar movement albumen provided by the invention and encoding gene thereof and the recombinant vectors that contains flagellar movement protein 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 flagellar movement albumen
(1) extraction and the purifying of the total DNA of basophilic Zymomonas mobilis (Alkalimonas amylolytica N10, CGMCC NO.0463)
Get basophilic Zymomonas mobilis (Alkalimonas amylolytica N10, CGMCC NO.0463) 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 flagellar movement protein gene
Analyze basophilic Zymomonas mobilis (Alkalimonas amylolytica N10, CGMCC NO.0463) after genomic information, the upstream and downstream primer of design flagellar movement protein gene, wherein upstream primer is 5 '-GCGGGATCCATGCGGTTTTTGTTGCC-3 ', and downstream primer is 5 '-CGCAGATCTTTAAGGTCTGTTCATCTGC-3 '.By the nucleic acid polymerase Pyrobest (Takara) of high-fidelity, with basophilic Zymomonas mobilis (Alkalimonas amylolytica N10, the CGMCC NO.0463) genome of said extracted, be the total length that template amplification goes out flagellar movement protein 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 876bp.
The gene function checking of embodiment 2 coding flagellar movement albumen
(1) structure of recombinant cloning vector pUC18-Aan10-motY
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-Aan10-motY 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-Aan10-motY) that recombinant vectors pUC18-Aan10-motY inserts, and confirm that the nucleotide sequence of extension increasing sequence that pUC18-Aan10-motY inserts and flagellar movement albumen is in full accord through order-checking.
(2) structure of e. coli k12 BW25113 flagellar movement protein delation body
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 flagellar movement protein gene of e. coli k12 BW2511, obtain the mutant E.coli K12 (Δ mot) that e. coli k12 BW25113 flagellar movement protein gene (Δ mot) lacks completely, flagellar movement protein gene is replaced by kanamycin gene.
(3) the alkali-proof mensuration of the e. coli k12 (pUC18-Aan10-motY) that recombinant vectors pUC18-Aan10-motY 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-Aan10-motY) that recombinant vectors pUC18-Aan10-motY 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 are parallel), result is as shown in Figure 1.
(4) (pUC18-Aan10-motY) alkali-proof mensuration of the e. coli k12 of flagellar movement protein delation (Δ mot) and the K12 (Δ mot) that imports recombinant vectors pUC18-Aan10-motY
By the K12 (Δ mot) that imports recombinant vectors pUC18-Aan10-motY (pUC18-Aan10-motY) and the e. coli k12 (Δ mot) of flagellar movement protein delation 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 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-Aan10-motY 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 flagellar movement protein gene (Aan10-motY) can improve the alkali resistance of e. coli k12.
As can be seen from Figure 2 the e. coli k12 (Δ mot) that, imports recombinant vectors pUC18-Aan10-motY 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 coli flagellum protein delation body K12 (Δ mot), this has also proved that the importing of flagellar movement protein gene (Aan10-motY) can improve the alkali resistance of e. coli k12 (Δ mot).
In sum, the flagellar movement protein gene that the application provides has important application potential at the aspects such as cultivation of Salt And Alkali Tolerance biology.By clone, obtain flagellar movement protein gene, and by genetically modified operation, microbe-derived flagellar movement albumen is imported in vegetable cell, the transfer-gen plant that obtains the raising of salt tolerant alkalescence becomes possibility.
Claims (7)
1. a flagellar movement albumen, is characterized in that, the aminoacid sequence of this flagellar movement albumen is as shown in SEQ ID No:2.
2. a flagellar movement protein 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. gene claimed in claim 2, recombinant vectors claimed in claim 4 have the application in alkali resistance genetically modified organism in cultivation, and described biology is intestinal bacteria.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN87104581A (en) * | 1986-12-24 | 1988-09-21 | 遗传学系统公司 | The monoclonal antibody of resisting pseudomonas aeruginosa flagellum |
| WO2006046017A2 (en) * | 2004-10-26 | 2006-05-04 | The Secretary Of State For Environment, Food & Rural Affairs | Vaccine and nucleic acids capable of protecting poultry against colonisation by campylobacter |
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2011
- 2011-10-31 CN CN201110338601.XA patent/CN103087160B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN87104581A (en) * | 1986-12-24 | 1988-09-21 | 遗传学系统公司 | The monoclonal antibody of resisting pseudomonas aeruginosa flagellum |
| WO2006046017A2 (en) * | 2004-10-26 | 2006-05-04 | The Secretary Of State For Environment, Food & Rural Affairs | Vaccine and nucleic acids capable of protecting poultry against colonisation by campylobacter |
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Granted publication date: 20140312 Termination date: 20191031 |