CN100351378C - Aldehyde-alcohol dehydrogenase gene - Google Patents
Aldehyde-alcohol dehydrogenase gene Download PDFInfo
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Abstract
The present invention relates to an aldehyde-alcohol dehydrogenase gene which belongs to the technical field of biological engineering. Because the cloning of the aldehyde-alcohol dehydrogenase gene is important to the metabolic engineering research of hydrogenogens B49 and the increase of hydrogen production quantity, the aldehyde-alcohol dehydrogenase gene is obtained by using the total DNA of highly effective hydrogenogens, namely EthanologenbacteriumhitB49, as a template and through PCR amplification. The full length of the aldehyde-alcohol dehydrogenase gene is 3098 bp, an initiator codon ATG is positioned at the 277 bp point, and a termination codon TAA is positioned at the 2889 bp point. The sequence does not contain introne, has 2616 bp of a complete open reading frame and can code 871 amino acids. The resources of the aldehyde-alcohol dehydrogenase gene are enlarged, and thus, a scientific basis is provided for the metabolic engineering research of Ethanologenbacterium hit B49 and the construction of gene engineering bacteria.
Description
Technical field
The invention belongs to technical field of bioengineering, be specifically related to a kind of aldehyde-alcohol dehydrogenase gene.
Background technology
Alcohol metabolism is the part of glucose metabolism, 1 molecule glucose is decomposed into 2 molecule pyruvic acid, 1 molecule pyruvic acid changes 1 molecule acetyl-CoA into, acetyl-CoA generates acetaldehyde under acetaldehyde dehydrogenase ALDH catalysis, be converted into ethanol by ethanol dehydrogenase ADH catalysis acetaldehyde again under NADH (Reduced nicotinamide-adenine dinucleotide) participates in.From the biological hydrogen production angle, produce the hydrogen hypothesis according to the NADH coupling, generate ethanol and can consume NADH, reduce hydrogen output; From the new forms of energy angle, ethanol is a kind of important industrial raw material, can make industrial alcohol, can do alcohol fuel, as the renewable energy resources to substituting and to alleviate the China's oil deficiency significant, be to accelerate exploitation substitute energy and new forms of energy, realize the effective measure of energy consumption structure multi-sourceization; Carrying out alcohol fuel can bring to country and stimulate agricultural development, improve the energy security system, reduce degree of dependence to oil, save foreign exchange, create more jobs, increase the state tax revenue, improve huge comprehensive income such as fuel qualities.Use ethanol as automobile fuel, can obviously reduce discharge of automobile exhaust fumes, effectively improve atmosphere quality.Studies show that the aflame C circulation of alcohol production and ethanol sealing, the CO of discharging
2Again absorbed by the alcohol production raw material, obviously slow down Global warming.In sum, no matter clone's aldehyde-alcohol dehydrogenase gene still from new forms of energy angle all is significant from the biological hydrogen production angle.
Hydrogenogens B49 is that doctor Lin Ming of Harbin Institute of Technology isolated plant height from the ethanol-type fermentation active sludge of biological hydrogen production reactor produces ethanolic hydrogen type fermentation strain, and Physiology and biochemistry and molecular biology identification result turn out to be a novel species or new the genus.This bacterial strain is in Chinese microorganism strain preservation center preservation, and deposit number is CGMCC1153.The high specific hydrogen-producing speed of hydrogenogens B49 is 25.0mmolH
2/ gdrycellh, unit volume hydrogen-producing speed are 1813.8mL/L-culture, and hydrogen production potential occupy international prostatitis.Its strain characteristics is the regular bacillus of unit cell growth; Peritrichous; G+; No pod membrane; No gemma; Obligate anaerobic; The circular bacterium colony of oyster white; Dai Shiwei 7.2h; At 28～43 ℃, grow under pH=3.3～8.5 conditions.The main tunning of B49 glycolysis glucose is ethanol, acetate, H
2, CO
2And lactic acid, the pathways metabolism of the novel organic waste water fermented type-ethanol-type fermentation that proposes referring to Ren Nanqi is inferred the pathways metabolism (see figure 1) of B49 glycolysis glucose.This shows that the clone of aldehyde-alcohol dehydrogenase gene is important to studying its metabolic engineering raising hydrogen output.
Summary of the invention
In view of improving hydrogen output to research hydrogenogens B49 metabolic engineering, the clone of aldehyde-alcohol dehydrogenase gene plays a very important role, the present invention is intended to separate aldehyde-alcohol dehydrogenase gene from Ethanologenbacterium hit B49 genome, enlarge the aldehyde-alcohol dehydrogenase gene resource, provide scientific basis thereby study and make up genetic engineering bacterium for the metabolic engineering of Ethanologenbacteriumhit B49.Aldehyde-alcohol dehydrogenase gene Badh of the present invention is to be template with the total DNA of highly effective hydrogen yield bacterium Ethanologenbacterium hit B49, obtains by pcr amplification.Aldehyde-alcohol dehydrogenase gene Badh full length gene 3098bp of the present invention, wherein A, C, G, T are respectively 797 (25.73%), 850 (27.44%), 744 (24.02%), 707 (22.82%).At the 277bp place initiator codon ATG is arranged, terminator codon TAA is arranged at the 2889bp place, at 204bp to there being a promoter sequence TTGCAACTTGCAAAAAAGTATCGTATAATAATATTGATAATATTTTAACA between the 249bp.This sequence intronless has the complete opening code-reading frame of 2616bp, 871 amino acid of encoding.The coded product molecular weight is 95.47kD, and iso-electric point pI is 6.42.By GenBank gene order compare of analysis, show with registered aldehyde-alcohol dehydrogenase homology be 52～65%, wherein the aldehyde-alcohol dehydrogenase gene homology with Bacillus licheniformis ATCC 14580 and Bacillus licheniformisDSM13 is the highest, be 65%, prove a new aldehyde-alcohol dehydrogenase gene, isolating aldehyde-alcohol dehydrogenase gene is connected with prokaryotic expression carrier pet-22b, be built into the pet-22b-ADH recombinant expression vector, after IPTG induces, give expression to the target protein of 100～120kD, confirm that this gene is a gene with expressive function.The present invention separates aldehyde-alcohol dehydrogenase gene from Ethanologenbacteriumhit B49 genome, enlarged the resource of aldehyde-alcohol dehydrogenase gene, provide scientific basis thereby study and make up genetic engineering bacterium for the metabolic engineering of Ethanologenbacterium hit B49.
Description of drawings
Fig. 1 is the pathways metabolism of hydrogenogens B49, and Fig. 2 is the structure collection of illustrative plates of pMD18-T carrier, and Fig. 3 is the restriction enzyme site structure iron of pMD18-T carrier, and Fig. 4 is TaKaRa LA PCR
TMThe pcr amplification schematic diagram of in vitro cloningKit test kit, Fig. 5 are Auele Specific Primer design attitude figure, and Fig. 6 is the structure collection of illustrative plates of pet-22b carrier, and Fig. 7 is the restriction enzyme site structure iron of pet-22b carrier.
Embodiment
Embodiment one: the described Ethanologenbacterium hit of present embodiment B49 aldehyde-alcohol dehydrogenase gene Badh is to be template with the total DNA of highly effective hydrogen yield bacterium Ethanologenbacterium hit B49, obtains by pcr amplification.Present embodiment has been carried out the prokaryotic expression detection to this gene, also this bacterial strain has been carried out the aldehyde-alcohol dehydrogenase vitality test simultaneously.Its concrete grammar is as follows:
1, the clone of aldehyde-alcohol dehydrogenase gene:
(1) PCR primer design:
According to the bacterium aldehyde-alcohol dehydrogenase protein sequence of including among the GenBank, concrete bacterial classification sees Table 1, through clustalw multisequencing compare of analysis, finds out the aminoacid sequence district (Blocks) of 10 no gap high conservatives.The protein sequence of these ten conservative regions is converted to the degenerate core nucleotide sequence, and calculates degeneracy, through having selected pair of degenerate primers for use, the sequence of this primer is referring to table 2.
The proteinic bacterial classification of the table 1 design used aldehyde-alcohol dehydrogenase of aldehyde-alcohol dehydrogenase gene degenerated primer
| Protein sequence | Bacterial classification |
| Aldehyde-alcohol dehydrogenase | Clostridium acetobutylicum ATCC824 |
| Aldehyde-alcohol dehydrogenase | Clostridium perfringens str：13 |
| Aldehyde-alcohol dehydrogenase | Clostridium tetani E88 |
| Aldehyde-alcohol dehydrogenase | Bacillus licheniformis ATCC 14580 |
| Aldehyde-alcohol dehydrogenase | Bacillus cereus ATCC 10987 |
| Aldehyde-alcohol dehydrogenase | Bacillus anthracis str：′Ames Ancestor |
| Aldehyde-alcohol dehydrogenase | Erwinia carotovora |
| Aldehyde-alcohol dehydrogenase | Escherichia coli K-12 W3110 |
| Aldehyde-alcohol dehydrogenase | Escherichia coli K-12 MG1655 |
| Aldehyde-alcohol dehydrogenase | Escherichia coli O157 EDL933 |
| Aldehyde-alcohol dehydrogenase | Photorhabdus luminescens |
| Aldehyde-alcohol dehydrogenase | Shigella flexneri 301 |
The degenerated primer of the amplification aldehyde-alcohol dehydrogenase that table 2. is selected for use
| The primer title | Primer sequence | Degeneracy |
| ADHJ1-forward (5 ' end primer) | 5’ACNAAYCCNACNWSNACNRCNATNTTYAAR3’ | 1048576 |
| ADHJ1-reverse (3 ' end primer) | 5’YKNGGRTTNGCNBYNGTRCAYTGRTC3’ | 98304 |
Annotate: N:A, C, T or G, Y:C or T, W:A or T, S:C or G, K:G or T, R:A or G, B: non-A.
(2) extraction of Ethanologenbacterium hit B49 genome DNA:
Get the about 50mL of Ethanologenbacterium hit B49 bacterium liquid, with magnificent Shun bacterial genomes DNA extraction agent box extraction in a small amount that Shanghai China Shun biotechnology company limited produces, extraction step is referring to a small amount of bacterial genomes DNA extraction agent box operational manual.
(3) the segmental pcr amplification of aldehyde-alcohol dehydrogenase gene part:
With the total DNA of Ethanologenbacterium hit B49 is that template is carried out pcr amplification.Reaction system: 1xBuffer, 0.6mM dNTP, each 1.0 μ M of primer ADHJ1-forward, ADHJ1-reverse, ExTaqDNA polysaccharase 2.5U, template DNA 0.2～1 μ g adds ddH
2O to 50 μ L.Response procedures: 95 ℃ of pre-sex change, 5min, 94 ℃, 30s; 55 ℃ drop to 35 ℃, fall 1 ℃, 40s at every turn; 72 ℃, 2min30s, totally 20 circulations; Press 94 ℃ then, 30s; 35 ℃, 40s; 72 ℃, 2min 30s, totally 23 circulations are extended 10min, 4 ℃ of preservations at 72 ℃ at last.
(4) clone of PCR product:
Pillar China Shun a small amount of glue that the PCR product is produced with Shanghai China Shun biotechnology company limited reclaims test kit and reclaims, and method reference reagent box is glue reclaimer operation handbook in a small amount.Reclaim product and be connected linked system with the pMD18-T cloning vector: pMD18-T carrier 1 μ l (50ng), (100～200ng), Ligation Solution5 μ l adds ddH to PCR product 2 μ l
2O to 10 μ l.16 ℃ of connections of spending the night.Connect product Transformed E .ColiDH5 α competent cell.Carry out blue hickie screening on the LB screening flat board of penbritin, X-gal and IPTG containing, and extract hickie bacterium colony plasmid and carry out bacterium colony PCR to identify positive recombinant.Wherein:
A, employing CaCl
2Legal system is equipped with E.coliDH5 α competent cell:
1. E.coliDH5 α streak culture 12～16h on the LB flat board, picking list bacterium colony insert and do not contain in the antibiotic LB liquid nutrient medium, and 37 ℃ of shaking culture are spent the night.
2. get the activatory E.coliDH5 α bacterium liquid 1ml that spends the night and place the fresh LB liquid nutrient medium of 100ml, 37 ℃ of shaking culture are to OD
600Be about 0.3.
3. bacterium liquid is sub-packed in the aseptic centrifuge tube of 50ml of two precoolings ice bath 30min.
4. 4 ℃, 4000rpm, centrifugal 10min abandons supernatant.
5. add the ice-cold 0.1M CaCl of 10ml
2, resuspended thalline, ice bath 30min.
6. 4 ℃, 4000rpm, centrifugal 10min abandons supernatant.
7. add the ice-cold 0.1M CaCl of 2ml
2, resuspended thalline.
B, heat shock method Transformed E .coliDH5 α competent cell:
1. get 200 μ l competent cells, place on ice, add 4 μ l and connect product, rotate the mixing content gently.
2. ice bath 30min.
3. heat shock 90s in 42 ℃ of water-baths does not shake.
4. ice bath 2～3min.
5. adding 800 μ l does not have additional antibiotic LB substratum, mixing, and 37 ℃ of pre-expression of 200～250rpm shaking table vibration are cultivated 45～60min.
6. room temperature, 4000rpm, centrifugal 5min discards 900 μ l supernatant liquors, and surplus liquid suspends thalline.
7. bacterium is coated on the LB solid medium of additional 50mg/LAmp, 4 μ l IPTG and 40 μ l X-gal.
8. dull and stereotypedly be placed to liquid in 37 ℃ of forwards and be absorbed, be inverted overnight incubation then.
The screening of c, positive recombinant and evaluation:
1. blue hickie reaction screening positive recombinant: according to the principle of α-Hu Bu reaction, the white colony that produces on the screening culture medium of additional X-gal and IPTG is the bacterium colony that has recombinant plasmid, blue colonies is the bacterium colony that has the recirculation carrier, but so positive recon of white colony preliminary evaluation.
2. the bacterium colony PCR of positive recombinant identifies: picking hickie bacterium colony, utilize universal primer RV-M and M13-47 to carry out PCR, the PCR condition: 95 ℃ of pre-sex change 10min; 94 ℃ of 30s, 50 ℃ of 30s, 72 ℃ of 90s, totally 35 circulations; 72 ℃ are extended 10min, 4 ℃ of preservations.Carrying out 0.8% agarose gel electrophoresis, if electrophoresis showed goes out the band with the target DNA size, prove that then the plasmid reorganization is correct, is positive recombinant.
The pMD18-T that adopts in the present embodiment is a kind of cloning vector commonly used, and size is 2692bp, has the Amp resistant gene; Regulating and controlling sequence and 146 amino acid whose coded messages of N-end of having one section intestinal bacteria lacZ, multiple clone site has been inserted in this coding region, if no foreign gene inserts, carrier can have complementary functions with the C-terminal sequence of intestinal bacteria lacZ, be the α complementation, producing has complete active beta-galactosidase enzymes; If there is foreign gene to insert, then destroyed its reading frame, produce the peptide section of the complementary ability of no α, therefore, white colony is the bacterium that has recombinant plasmid on the screening culture medium of additional X-gal and IPTG, blue colonies is the bacterium that has the recirculation carrier.The described pMD18-T carrier of present embodiment is available from Dalian Bao Bio-Engineering Company, and its pMD18-T plasmid map and restriction enzyme site thereof are seen Fig. 2 and 3.
(5) determined dna sequence and analysis:
Positive recombinant selects for use the handsome Bioisystech Co., Ltd in Invitrogen Shanghai automatic sequence analyser to carry out sequencing, and the sequence homology comparison is carried out with blast program on http://www.ncbi.nlm.gov website.
(6) design of primers of clone's aldehyde-alcohol dehydrogenase gene part fragment both sides sequence C assette PCR:
Adopt TaKaRa LA PCR
TMIn vitro cloning Kit test kit clone aldehyde-alcohol dehydrogenase gene fragment both sides sequence.Based on the aldehyde-alcohol dehydrogenase gene fragment sequence of having cloned, Cassette primer C1 in design and the test kit, the upstream primer ADH-S11-lower that Cassette primer C2 primer mates respectively, ADH-S12-lower and downstream primer ADH-S21-upper, ADH-S22-upper, concrete sequence sees Table 3.
The Cassette PCR primer of table 3. amplification aldehyde-alcohol dehydrogenase gene part fragment both sides sequence
| The primer title | Primer sequence |
| Cassette primer C1 | 5’GTACATATTGTCGTTAGAACGCGTAATACGACTCA3’ |
| Cassette primer C2 | 5’CGTTAGAACGCGTAATACGACTCACTATAGGGAGA3’ |
| ADH-S11-lower(C1) | 5’GCTATCGTTGACCCGCAGTTTGTTATGAGG3’ |
| ADH-S12-lower(C2) | 5’GATCTGGCCGAAATACGAGACTTATCTTGCCC3’ |
| ADH-S21-upper(C1) | 5’GTATTTCATGATGGATGTCGTTATCGGTGGAGTGG3’ |
| ADH-S22-upper(C2) | 5’GGTTTTGACGGAGATGAGGGATTTAAAGATAGCGG3’ |
Wherein, the principle of Cassette PCR and design of primers require as follows:
A, Cassette PCR principle: concrete principle is seen Fig. 4.Because in design, 5 ' end of Cassette does not have phosphate, so the 3 ' 5 ' terminal connecting portion terminal and Cassette of Target DNA forms breach.At first circulation time of the PCR reaction first time, the extension that begins from Primer C1 stops at connecting portion, has limited the amplification between Primer C1 and the same primer of Primer C1, thereby has controlled non-specific pcr amplification.Have only from Primer S11 or Primer S21 to begin to extend synthetic DNA chain, just can become the template of Primer C1, carry out the specificity extension self-increasing reaction of DNA.Use inboard Primer (Primer C2, Primer S12 or S22) further to carry out the PCR reaction second time again, target DNA specifically can efficiently increase.
When proteinic aminoacid sequence is known, can design Mixed primer according to Given information, the proteinic cDNA of amplification coding.
The concrete operations step of this test kit is poly-as follows:
1) with EcoR I or BamH I restriction enzyme Ethanologenbacterium hit B49 genomic dna is decomposed fully.
2) carry out ligation with Cassette joint with EcoR I or BamH I restriction enzyme restriction enzyme site.
3) with Cassette Primer (Primer C1) with according to the Primer (Primer S11 or Primer S21) of the dna sequence dna of known region design, carry out the 1st PCR (1st PCR) reaction.
4) get 3) the part of PCR reaction solution make template, use inboard Primer (Primer C2 and Primer S12 or Primer S22) to carry out the 2nd PCR (2nd PCR) reaction, target DNA fragment specifically increases.
The standard of b, Specific Primer (S11 or S21, S12 or S22):
1) according to known region design Primer (see figure 5).Design direction is the direction that needs the zone of ignorance of amplification.The inboard at S1 should be designed in the position of S2, and the distance between two primers does not have strict regulation.
Design also should be noted that during primer following some:
1. primer length is that 20～35mer (is preferably 30～35mer) during amplification long-chain DNA.
2. GC content avoids local GC or AT to concentrate about 50%.Particularly AT is unconcentrated for 3 of primer ' end.
3. primer self does not form tangible secondary structures such as hair clip.
4. two Specific Primer (S11 or S21, S12 or S22) will be used in combination with Cassette Primer (C1, C2), so design the time also will be considered to form primer dimer with the paired primer, particularly 3,4 bases of 3 ' end not with the complementation of paired primer sequence.
Should note when 2) designing primer according to proteinic aminoacid sequence following some:
1. at first aminoacid sequence is converted to the base sequence of coded amino acid.Should select the few zone design primer of degeneracy, but few with degeneracy and short primer compares, the many and long primer of degeneracy is proper not as good as using as far as possible.
If 2. want to reduce the primer degeneracy, can consider that Codon usage designs.
3. 3 ' end is not wanted degeneracy.
If when 4. using Mixed primer to carry out pcr amplification, annealing temperature need be reduced, and its result tends to cause nonspecific amplification.If the relative dna sequence information more for a long time, can further design a primer again, in order to identify target DNA fragment in the inboard.
(7) the Cassette pcr amplification of clone's aldehyde-alcohol dehydrogenase gene part fragment both sides sequence:
According to the aldehyde-alcohol dehydrogenase gene fragment sequence of having cloned, analyze the restriction endonuclease sites situation of this sequence, find out the restriction enzyme that this sequence does not have, and TaKaRa LA PCR
TMThe restriction enzyme that this enzyme joint is arranged in the in vitro cloningKit test kit, detailed process is as follows: the amplification in aldehyde-alcohol dehydrogenase downstream: select EcoR I digestion with restriction enzyme B49 genomic dna for use, the genomic dna after enzyme is cut is connected 3h with EcoR I joint in 16 ℃; The amplification in aldehyde-alcohol dehydrogenase downstream: select for use BamH I enzyme to cut the B49 genomic dna, the genomic dna after enzyme is cut is connected 3h with Sau3A I joint in 16 ℃.CassettePCR reaction system and reaction conditions are as follows:
The restriction enzyme endonuclease reaction of A, DNA: reaction system sees Table 4.
Table 4
| Genomic dna | 5μg |
| EcoR I restriction enzyme | 40U * |
| 10 * restriction enzyme Buffer | 5μl |
| Sterile purified water | up to 50μl |
*The necessary enzyme amount that representative is decomposed DNA is fully decided according to the DNA purity of preparation, and general 10U decomposes 1 μ g DNA.
B, ligation:
1. by following component preparation ligation liquid (table 5).
Table 5
| EcoR I enzyme is cut genomic DNA fragment | 5μl |
| EcoR I Cassette joint | 2.5μl |
| Ligation Solution I | 15μl |
| Ligation Solution II | 7.5μl |
2. 16 ℃ were reacted 30 minutes.
3. after reaction finishes, carry out ethanol sedimentation and reclaim DNA.
4. be dissolved in the sterile purified water of 5 μ l.
C, pcr amplification:
1. with B-4. dna solution 1 μ l join in the 33.5 μ l sterile purified waters, 94 ℃ the heating 10 minutes.
2. by following component preparation (1st) PCR reaction solution (table 6) first time.
Table 6
| C- | 34.5μl |
| 10×LA PCR Buffer II(Mg 2+ plus) | 5μl |
| TaKaRa LA Taq | 0.5μl |
| dNTP Mixture | 8μl |
| Primer C1 | 1μl |
| Primer S11 or S21 | 1μl |
3. carry out the 1stPCR reaction by following condition.
94 ℃ of 30sec, 55 ℃ of 2min, 72 ℃ of 1min, totally 30 circulations.
4. with aqua sterilisa B-PCR reaction solution is 3. diluted (1～10,000 times of dilution) by suitable multiple, get 1 μ l again and carry out (for the second time) 2nd PCR reaction, 2nd PCR reaction solution component such as table 7.
Table 7
| B-1stPCR reaction solution (or diluent) 4. | 1μl |
| 10×LA PCR Buffer II(Mg 2+ plus) | 5μl |
| TaKaRa LA Taq | 0.5μl |
| dNTP Mixture | 8μl |
| Primer C2 | 1μl |
| Primer S12 or S22 | 1μl |
| Sterile purified water | up to 50μl |
5. carry out 2nd PCR reaction by following condition.
94 ℃ of 30sec, 55 ℃ of 2min, 72 ℃ of 1min circulate 30 times altogether.
6. after reaction finishes, get PCR reaction solution (5～10 μ l) and carry out agarose gel electrophoresis, the PCR fragment that can obtain increasing.
(8) pcr amplification of aldehyde-alcohol dehydrogenase full-length gene, clone and order-checking:
According to the sequences Design of having spliced total length checking primer ADHWU-Forward, ADHWU-Reverse, ADHWL-Forward and ADHWL-Reverse (seeing Table 8), be template amplification total length aldehyde-alcohol dehydrogenase gene with Ethanologenbacterium hit B49 genome DNA.The PCR reaction system is 94 ℃ of 5min, 94 ℃ of 30s, and 66 ℃ of 30s, 72 ℃ of 1min30s, totally 35 circulations, last 72 ℃ are extended 10min.
Table 8. aldehyde-alcohol dehydrogenase gene total length checking primer
| Amplification region | The primer title | Primer sequence |
| 36-2154 | ADHWU-Forward | 5’GCCCGTGTTAAAACGAGAAAATGTTTCCCTTTCCGTCATCCG3’ |
| ADHWU-Reverse | 5’GAAGGAATATTGGTGTTGTAGATATCGCCGATACCGCCC3’ | |
| 1715-3706 | ADHWL-Forward | 5’GCAAACGGCTGCTACTTCACCAATCCGGAAGAAACGGCC3’ |
| ADHWL-Reverse | 5’ACCAATACGATTATTATTATGTTCCCACTTTTTACGTAGACGG3’ |
(9) expression of aldehyde-alcohol dehydrogenase gene:
With chitinase gene Back and prokaryotic expression carrier pet-22b reorganization, be built into recombinant expression plasmid pet-22b-Back, the heat shock method is transformed in prokaryotic expression host bacterium E.coliBL21 (DE3) competent cell, 1.0mM after IPTG induced 3.5h, the SDS-PAGE gel electrophoresis through 12%, coomassie brilliant blue staining detected the expression of gene situation.The pet-22b carrier is purchased the company in Novagen, and its plasmid map and restriction enzyme site are seen Fig. 6 and 7.
Multiple clone site sequence according to the pet-22b expression vector, locate to introduce BamH I restriction enzyme site at the 253bp of aldehyde-alcohol dehydrogenase gene (initiator codon ATG), locate to introduce Xho I restriction enzyme site at 1450bp (terminator codon TAA), concrete primer sees Table 9.PCR reaction conditions: 95 ℃ of pre-sex change 5min; 94 ℃ of sex change 30s, 65 ℃ of annealing 30s, 72 ℃ are extended 2min30s, totally 35 circulations; Last 72 ℃ are extended 10min.Glue reclaims the PCR product, cuts through BamH I and Xho I enzyme, reclaims at glue, is connected 3h with the pet-22b prokaryotic expression carrier that BamH I cuts with the XhoI enzyme and glue reclaims in 16 ℃ with same.Should connect product Transformed E .ColiDH5 α competent cell.Carry out blue hickie screening on the LB screening flat board of penbritin, X-gal and IPTG containing, and extract hickie bacterium colony plasmid and carry out bacterium colony PCR to identify positive recombinant.Extract the positive colony plasmid, this plasmid is transformed BL21 (DE3) plys competent cell.Carry out blue hickie screening on the LB screening flat board of penbritin, X-gal and IPTG containing, and extract hickie bacterium colony plasmid and carry out bacterium colony PCR to identify positive recombinant.
Table 9. aldehyde-alcohol dehydrogenase gene is expressed and is used primer
| The primer title | Primer sequence |
| ADH-22b-forward | 5’AGGGGGCggatccATGGCAACCGAAACAAAAGAAGTCGCCCAGG3’ |
| ADH-22b-reverse | 5’GGTAAACCTCGAGTTTGCCGTAATAAGCGGCTTTATAGAGCTCGG3’ |
2, Ethanologenbacterium hit B49 E.C. 2.7.2.1 vitality test:
(1), measuring method:
It is by measuring ethanol dehydrogenase reduction NAD that alcohol dehydrogenase activity is measured
+The amount of one-tenth NADH is calculated.
Reaction solution: 900 μ l, 12mM trisodium phosphate (pH8.5); 50 μ l1,5mMNAD
+30 μ l cellular enzymes crude extract, totally 980 μ l.Add 20 μ l dehydrated alcohols at last and begin reaction.At visible spectrophotometer 340nm place reading.
(2), protein content determination:
Adopt Shanghai China Shun biotechnology BCA of company limited protein content determination test kit.
(3), the definition (U) of enzyme unit alive:
Micromole's number of every milligram of (milligram) albumen per minute reductive NADH product.The aldehyde-alcohol dehydrogenase vigor that present embodiment obtains is 48U.
The described Ethanologenbacterium hit of present embodiment B49 aldehyde-alcohol dehydrogenase gene Badh is an aldehyde-alcohol dehydrogenase, registered sequence there are differences among this gene of Blast homology comparison result shows of sequence and the GenBank, is a new gene.
The Badh gene sequence characteristic: this full length gene 3098bp, wherein A, C, G, T are respectively 797 (25.73%), 850 (27.44%), 744 (24.02%), 707 (22.82%).At the 277bp place initiator codon ATG is arranged, terminator codon TAA is arranged, at the promoter sequence TTGCAACTTGCAAAAAAGTATCGTATAATAATATT GATAATATTTTAACA of 204bp to a supposition of existence between the 249bp at the 2889bp place.This sequence intronless has the complete opening code-reading frame of 2616bp, 871 amino acid of encoding.The coded product molecular weight is 95.47kD, and iso-electric point pI is 6.42.
Sequence table
＜110〉Harbin Institute of Technology
＜120〉aldehyde-alcohol dehydrogenase gene
<160>1
<210>1
<211>3098
<212>DNA
＜213〉aldehyde-alcohol dehydrogenase gene and coded product sequence thereof
<400>1
1 GTGTACTCGTCGAAAACGTGCACCTGAGTGACAGCCTGATGACATTGTGCTACATGGTAA
61 GGGATTTATCCGAAAAAGAAACACGGGACAATTGAACTGCTTTGATACCGTCATGACCAT
121 TCGGCTCGTTGCACGAAAACGACAAATCAAACGGGACATGACGTCATCGTTTTATAATAT
181 TTGACAAATTGCACAGCATTCACTTGCAACTTGCAAAAAAGTATCGTATAATAATATTGA
241 TAATATTTTAACAATATTGCTTAAGGGGGCAATTTGATGGCAACCGAAACAAAAGAAGTC
M A T E T K E V
301 GCCCAGGAAGCGCAGAAACCCGACGTCAAACAAATGATTGACGAACTGGTGGCAAAAGCG
A Q E A Q K P D V K Q M I D E L V A K A
361 CAGAAAGCTCTGGACGAGTTTTCTACCTTCAACCAGGAGCAGGTTGACAAAATCGTCCAT
Q K A L D E F S T F N Q E Q V D K I V H
421 GCCATGGCACTTGCGGCGCTGGATAAGCATATGTATCTGGCTAAACTCGCCGTGGAAGAA
A M A L A A L D K H M Y L A K L A V E E
481 ACCGGACGCGGTATTTACGAAGACAAGATTACCAAAAACCTGTACGCAAGCGAATATATC
T G R G I Y E D K I T K N L Y A S E Y I
541 TGGCATGACATCAAATATGCAAAGACCGTCGGCGTCATCGACGAAAACGAGATGGAAGAA
W H D I K Y A K T V G V I D E N E M E E
601 TATGTGGATATCGCCGAGCCTGTCGGTATCATCGCGGGCGTTACCCCGGTAACCAACCCC
Y V D I A E P V G I I A G V T P V T N P
661 ACCTCCACCGCTATCTTTAAATCCCTCATCTCCGTCAAAACCCGCAACCCCATCATTTTC
T S T A I F K S L I S V K T R N P I I F
721 GGTTTCCATCCCGCTGCCCAGAAATGCTGCGCGGAAACGGCCCGCATTCTGAATGAGGCG
G F H P A A Q K C C A E T A R I L N E A
781 GCTGTGGCTGCCGGTGCCCCCGAAAACATTGTGCAGTTCATCCCCCATCCTTCCATCGAA
A V A A G A P E N I V Q F I P H P S I E
841 GCGACCAATGCGTTGATGAATCATCCCGGTGTCGCCACGATTTTGGCGACCGGCGGCCCC
A T N A L M N H P G V A T I L A T G G P
901 GGCATGGTCAAGGCTGCGTATTCCTGCGGCAAACCGGCGCTGGGTGTCGGTCCCGGCAAC
G M V K A A Y S C G K P A L G V G P G N
961 GTCCCCTGCTATGTCGAAAAAACAGCGAAACTCCGCCGTGCCTGCCATGACCTCATCCTT
V P C Y V E K T A K L R R A C H D L I L
1021 TCCAAAACGTTCGACAACGGCATGATCTGCGCTTCCGAACAGGCTGCGATTGTGGATAAA
S K T F D N G M I C A S E Q A A I V D K
1081 GAAATCGCGGCGGATTTTGAAAAGATCATGAAAGCAAACGGCTGCTACTTCACCAATCCG
E I A A D F E K I M K A N G C Y F T N P
1141 GAAGAAACGGCCAAACTCGGCAAATATGTGATCACCGAGAAAATGAGCGTGAATCCCCCG
E E T A K L G K Y V I T E K M S V N P P
1201 GTCGTCGGCCAGTCCGCTGTCTGGATCGCCGAGCAGGCCGGCATCAAAGTACCTGCCGAC
V V G Q S A V W I A E Q A G I K V P A D
1261 ACCAAGGTCATTCTTGCCAAACTTGACAAGGTCGATTTTGATGTGCCGCTGGCTCACGAA
T K V I L A K L D K V D F D V P L A H E
1321 AAACTTTCTCCGGTGCTTGGTTATTATATCGCCAACAGCACGGAGGATGCTTTCAAAGCT
K L S P V L G Y Y I A N S T E D A F K A
1381 GCGCTTCGCATGCTGGAAATCGGCGGTCTGGGTCACTCTGCAGCCATCCACTCCACCGAT
A L R M L E I G G L G H S A A I H S T D
1441 AACGACATCATCATGAAATACGGCGAAGCCATGAAGGTCGGCCGTGTGCTGGTCAACAGC
N D I I M K Y G E A M K V G R V L V N S
1501 CCGTCTTCTCAGGGCGGTATCGGCGATATCTACAACACCAATATTCCTTCCCTTACCCTT
P S S Q G G I G D I Y N T N I P S L T L
1561 GGCTGCGGCTCCTATGGCCACAACTCTGTTTCGCAGAACGTCAGTGCGGTCAACCTGATC
G C G S Y G H N S V S Q N V S A V N L I
1621 AACAAAAAGCGCATTGCCAAGAGGAGAGTCAACATGCAGTGGTTTAAAGTGCCGCCGAAG
N K K R I A K R R V N M Q W F K V P P K
1681 ATTTACTTCGAGTTCGATGCGATTCAGTACCTGGAGAAAATGCCGAACATCTCCCGTGCA
I Y F E F D A I Q Y L E K M P N I S R A
1741 TTCATCGTCACCGACCCTGTAATGGTCAAGCTCGGAAATGTGGATAAGGTTCTTTATTAT
F I V T D P V M V K L G N V D K V L Y Y
1801 CTCCGCAAACGCAAAGAGTATTGCCACAGTGAGATTTTCTCCGAAGTTGAGCCCGACCCG
L R K R K E Y C H S E I F S E V E P D P
1861 TCTTTTGAAACGGTTATGCGCGGCGTGGAAATGATGAAGAAATTTGAGCCGGATGTCATC
S F E T V M R G V E M M K K F E P D V I
1921 ATCGCACTCGGCGGCGGTTCTTCCATGGATGCCGCGAAGGGCATGTGGCTCTTCTATGAG
I A L G G G S S M D A A K G M W L F Y E
1981 CATCCGGACTTTGATTTCAAAGGCGCACATGAGAAATTCATGGATATCCGCAAGCGCACT
H P D F D F K G A H E K F M D I R K R T
2041 TATCACTTCCCCGAACTGGGCCAGAAATGCCAGATGGTCGCCGTGCCGACTACTTCCGGC
Y H F P E L G Q K C Q M V A V P T T S G
2101 ACCGGCTCCGAAGTGACGTCTTTTGCGGTCATCACCGACAAGGTCAATAACTTCAAATAT
T G S E V T S F A V I T D K V N N F K Y
2161 CCGCTCGCCGACTATGCGCTCACTCCGAATGTCGCTATCGTTGACCCGCAGTTTGTTATG
P L A D Y A L T P N V A I V D P Q F V M
2221 ACCATGCCGAAATCCACCACGGCGGATACCGGTTTGGACGTGCTGACCCACGCCATTGAA
T M P K S T T A D T G L D V L T H A I E
2281 GCCTATGTCTCCGTTTTGGCAAACGATTACACTGATGGTCTGGCCCTGAAAGCGATTGAG
A Y V S V L A N D Y T D G L A L K A I E
2341 ATCGTCTTCAAATATCTGCCCCGTGCCTATGCGGACGGTGCCAAAGACGGCGAAGCGCGC
I V F K Y L P R A Y A D G A K D G E A R
2401 ATGAAAATGCACAACGCTTCCTGCATCGCCGGTATGGCGTTCAGCAATGCGTTCCTTGGC
M K M H N A S C I A G M A F S N A F L G
2461 TTGAACCACTCCATGGCGCACAAACTGGGCGGCGAATACCACATTCCGCACGGTCGTGCC
L N H S M A H K L G G E Y H I P H G R A
2521 AACGCCATTCTGCTTCCCTATGTCGTTGAATACAACGGCAATCCGAAGCCGACCAAATAT
N A I L L P Y V V E Y N G N P K P T K Y
2581 GCGATCTGGCCGAAATACGAGACTTATCTTGCCCCGCAGCGTTTCCAAGAGATTGCCCGC
A I W P K Y E T Y L A P Q R F Q E I A R
2641 CATGTCGGCCTGAAGGCAAGCACACCGGAAGAGGGCGTTGCTTCCCTCGTGCAGGCGATT
H V G L K A S T P E E G V A S L V Q A I
2701 CGCGATTTGATGAAGACCGTCAATGAGCCGATGAGCATTCAAGCCACCGGCGTGCCCGAG
R D L M K T V N E P M S I Q A T G V P E
2761 AACGTGTTCCTCACCAATCTGGATTCTCTGGCTGATAAAGCGTTTTCCGACCAGTGCACC
N V F L T N L D S L A D K A F S D Q C T
2821 GGCGCAAATCCGCGTCTGCCGCTCGTCAGCGAGATCGCCGAGCTCTATAAAGCCGCTTAT
G A N P R L P L V S E I A E L Y K A A Y
2881 TACGGCAAATAAACTGTTTACCAAGCGCACAATAAAAAAGGGGCCTGCTTTTTGCAGGTC
Y G K
2941 CCTTTTTTATTGTGCGCCACTTCAGCAATCCGTGCCAAACAGAAAATTTCATCCTTTTAC
3001 GGCCGCTTCGAATACCGCTTCGATTTTTTCCGGACTCGGTATTCCCTCGTGTAGCTTATT
3061 TCCGTCTACGTAAAAAGTGGGAACATAATAATAATCGT
Claims (1)
1, aldehyde-alcohol dehydrogenase gene is characterized in that the gene order of described aldehyde-alcohol dehydrogenase gene is:
GTGTACTCGTCGAAAACGTGCACCTGAGTGACAGCCTGATGACATTGTG
CTACATGGTAAGGGATTTATCCGAAAAAGAAACACGGGACAATTGAACT
GCTTTGATACCGTCATGACCATTCGGCTCGTTGCACGAAAACGACAAATC
AAACGGGACATGACGTCATCGTTTTATAATATTTGACAAATTGCACAGCAT
TCACTTGCAACTTGCAAAAAAGTATCGTATAATAATATTGATATATTTTTAA
CAATATTGCTTAAGGGGGCAATTTGATGGCAACCGAAACAAAAGAAGTC
GCCCAGGAAGCGCAGAAACCCGACGTCAAACAAATGATTGACGAACTG
GTGGCAAAAGCGCAGAAAGCTCTGGACGAGTTTTCTACCTTCAACCAGG
AGCAGGTTGACAAAATCGTCCATGCCATGGCACTTGCGGCGCTGGATAA
GCATATGTATCTGGCTAAACTCGCCGTGGAAGAAACCGGACGCGGTATTT
ACGAAGACAAGATTACCAAAAACCTGTACGCAAGCGAATATATCTGGCAT
GACATCAAATATGCAAAGACCGTCGGCGTCATCGACGAAAACGAGATGG
AAGAATATGTGGATATCGCCGAGCCTGTCGGTATCATCGCGGGCGTTACC
CCGGTAACCAACCCCACCTCCACCGCTATCTTTAAATCCCTCATCTCCGTC
AAAACCCGCAACCCCATCATTTTCGGTTTCCATCCCGCTGCCCAGAAATG
CTGCGCGGAAACGGCCCGCATTCTGAATGAGGCGGCTGTGGCTGCCGGT
GCCCCCGAAAACATTGTGCAGTTCATCCCCCATCCTTCCATCGAAGCGAC
CAATGCGTTGATGAATCATCCCGGTGTCGCCACGATTTTGGCGACCGGCG
GCCCCGGCATGGTCAAGGCTGCGTATTCCTGCGGCAAACCGGCGCTGGG
TGTCGGTCCCGGCAACGTCCCCTGCTATGTCGAAAAAACAGCGAAACTC
CGCCGTGCCTGCCATGACCTCATCCTTTCCAAAACGTTCGACAACGGCAT
GATCTGCGCTTCCGAACAGGCTGCGATTGTGGATAAAGAAATCGCGGCG
GATTTTGAAAAGATCATGAAAGCAAACGGCTGCTACTTCACCAATCCGG
AAGAAACGGCCAAACTCGGCAAATATGTGATCACCGAGAAAATGAGCGT
GAATCCCCCGGTCGTCGGCCAGTCCGCTGTCTGGATCGCCGAGCAGGCC
GGCATCAAAGTACCTGCCGACACCAAGGTCATTCTTGCCAAACTTGACA
AGGTCGATTTTGATGTGCCGCTGGCTCACGAAAAACTTTCTCCGGTGCTT
GGTTATATATCGCCAACAGCACGGAGGATGCTTTCAAAGCTGCGCTTCG
CATGCTGGAAATCGGCGGTCTGGGTCACTCTGCAGCCATCCACTCCACC
GATAACGACATCATCATGAAATACGGCGAAGCCATGAAGGTCGGCCGTG
TGCTGGTCAACAGCCCGTCTTCTCAGGGCGGTATCGGCGATATCTACAAC
ACCAATATTCCTTCCCTTACCCTTGGCTGCGGCTCCTATGGCCACAACTCT
GTTTCGCAGAACGTCAGTGCGGTCAACCTGATCAACAAAAAGCGCATTG
CCAAGAGGAGAGTCAACATGCAGTGGTTTAAAGTGCCGCCGAAGATTTA
CTTCGAGTTCGATGCGATTCAGTACCTGGAGAAAATGCCGAACATCTCCC
GTGCATTCATCGTCACCGACCCTGTAATGGTCAAGCTCGGAAATGTGGAT
AAGGTTCTTTATTATCTCCGCAAACGCAAAGAGTATTGCCACAGTGAGAT
TTTCTCCGAAGTTGAGCCCGACCCGTCTTTTGAAACGGTTATGCGCGGCG
TGGAAATGATGAAGAAATTTGAGCCGGATGTCATCATCGCACTCGGCGG
CGGTTCTTCCATGGATGCCGCGAAGGGCATGTGGCTCTTCTATGAGCATC
CGGACTTTGATTTCAAAGGCGCACATGAGAAATTCATGGATATCCGCAAG
CGCACTTATCACTTCCCCGAACTGGGCCAGAAATGCCAGATGGTCGCCG
TGCCGACTACTTCCGGCACCGGCTCCGAAGTGACGTCTTTTGCGGTCATC
ACCGACAAGGTCAATAACTTCAAATATCCGCTCGCCGACTATGCGCTCAC
TCCGAATGTCGCTATCGTTGACCCGCAGTTTGTTATGACCATGCCGAAAT
CCACCACGGCGGATACCGGTTTGGACGTGCTGACCCACGCCATTGAAGC
CTATGTCTCCGTTTTGGCAAACGATTACACTGATGGTCTGGCCCTGAAAG
CGATTGAGATCGTCTTCAAATATCTGCCCCGTGCCTATGCGGACGGTGCC
AAAGACGGCGAAGCGCGCATGAAAATGCACAACGCTTCCTGCATCGCCG
GTATGGCGTTCAGCAATGCGTTCCTTGGCTTGAACCACTCCATGGCGCAC
AAACTGGGCGGCGAATACCACATTCCGCACGGTCGTGCCAACGCCATTC
TGCTTCCCTATGTCGTTGAATACAACGGCAATCCGAAGCCGACCAAATAT
GCGATCTGGCCGAAATACGAGACTTATCTTGCCCCGCAGCGTTTCCAAGA
GATTGCCCGCCATGTCGGCCTGAAGGCAAGCACACCGGAAGAGGGCGT
TGCTTCCCTCGTGCAGGCGATTCGCGATTTGATGAAGACCGTCAATGAGC
CGATGAGCATTCAAGCCACCGGCGTGCCCGAGAACGTGTTCCTCACCAA
TCTGGATTCTCTGGCTGATAAAGCGTTTTCCGACCAGTGCACCGGCGCAA
ATCCGCGTCTGCCGCTCGTCAGCGAGATCGCCGAGCTCTATAAAGCCGCT
TATTACGGCAAATAAACTGTTTACCAAGCGCACAATAAAAAAGGGGCCT
GCTTTTTGCAGGTCCCTTTTTTATTGTGCGCCACTTCAGCAATCCGTGCCA
AACAGAAAATTTCATCCTTTTACGGCCGCTTCGAATACCGCTTCGATTTTT
TCCGGACTCGGTATTCCCTCGTGTAGCTTATTTCCGTCTACGTAAAAAGTG
GGAACATAATAATAATCGT。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2006100096156A CN100351378C (en) | 2006-01-12 | 2006-01-12 | Aldehyde-alcohol dehydrogenase gene |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2006100096156A CN100351378C (en) | 2006-01-12 | 2006-01-12 | Aldehyde-alcohol dehydrogenase gene |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1807640A CN1807640A (en) | 2006-07-26 |
| CN100351378C true CN100351378C (en) | 2007-11-28 |
Family
ID=36839733
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB2006100096156A Expired - Fee Related CN100351378C (en) | 2006-01-12 | 2006-01-12 | Aldehyde-alcohol dehydrogenase gene |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN100351378C (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111689649A (en) * | 2020-06-19 | 2020-09-22 | 安徽国星生物化学有限公司 | Treatment method of acetaldehyde wastewater |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5482846A (en) * | 1988-08-31 | 1996-01-09 | University Of Florida | Ethanol production in Gram-positive microbes |
| CN1600862A (en) * | 2003-09-23 | 2005-03-30 | 中国农业科学院生物技术研究所 | Clone and application of alcohol fermentation gene of unit cell fungus through motion ferment |
| CN1644679A (en) * | 2004-08-27 | 2005-07-27 | 哈尔滨工业大学 | Hydrogen generating bacteria and its screening method |
-
2006
- 2006-01-12 CN CNB2006100096156A patent/CN100351378C/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5482846A (en) * | 1988-08-31 | 1996-01-09 | University Of Florida | Ethanol production in Gram-positive microbes |
| CN1600862A (en) * | 2003-09-23 | 2005-03-30 | 中国农业科学院生物技术研究所 | Clone and application of alcohol fermentation gene of unit cell fungus through motion ferment |
| CN1644679A (en) * | 2004-08-27 | 2005-07-27 | 哈尔滨工业大学 | Hydrogen generating bacteria and its screening method |
Non-Patent Citations (3)
| Title |
|---|
| 发酵条件对发酵产氢细菌B49产氢的影响 王相晶等.太阳能学报，第26卷第1期 2005 * |
| 影响产氢发酵细菌B49产氢的部分因子研究 王相晶等.东北农业大学学报，第36卷第5期 2005 * |
| 高效产氢新菌种的分离鉴定与16S rDNA全序列 李永峰等.哈尔滨工业大学学报，第37卷第3期 2005 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1807640A (en) | 2006-07-26 |
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