CN1321185C - Acetokinase gene - Google Patents

Abstract

The present invention relates to an acetokinase gene which belongs to the field of biological engineering. Because the clone of the acetokinase gene is important to the research of hydrogenogens B49 metabolic engineering and the increase of hydrogen production quantity, the present invention aims to separate the acetokinase gene from the Ethanologenbacterium hit B49 genome. The acetokinase gene Back of the present invention is obtained with the total DNA of Ethanologenbacterium hit B49 as a template by PCR amplification, and has expressive function. The full length of the gene is 2034 bp, the gene does not contain intron, has 1200 bp of complete open reading frame and can code 399 amino acids. The present invention enlarges the resources of the acetokinase gene, and thus, a scientific basis is provided for the metabolic engineering research of Ethanologenbacterium hit B49 and the construction of gene engineering bacteria.

Description

Acetokinase gene

Technical field

The invention belongs to bioengineering field, relate to a kind of Acetokinase gene.

Background technology

Acetate 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 need be undertaken by the priority catalysis of phosphotransacetylase PTA and E.C. 2.7.2.1 AK: acetyl-CoA generates acetylphosphate under phosphotransacetylase PTA catalysis, under participating in, ATP is converted into acetate again by E.C. 2.7.2.1 AK catalysis acetylphosphate, and the acetate that generates excretes, cause the accumulation of acetate in the substratum, reduce the pH value of substratum, thereby suppress the growth of bacterium.Studies show that: the variation of pH can change the fermented type of microorganism, thereby influences H ₂Output.

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 25.0mmolH2/gdrycellh, and the unit volume hydrogen-producing speed is 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 Acetokinase gene is important to studying its metabolic engineering raising hydrogen output.

Summary of the invention

Play a very important role in view of the clone of Acetokinase gene improves hydrogen output to research hydrogenogens B49 metabolic engineering, the present invention is intended to separating acetic acid kinase gene from Ethanologenbacterium hit B49 genome.Ethanologenbacterium hit B49 Acetokinase gene Back of the present invention is to be template with the total DNA of highly effective hydrogen yield bacterium Ethanotogenbacterium hit B49, obtains by pcr amplification.Ethanologenbacterium hit B49 Acetokinase gene Back sequence total length 2034bp of the present invention, wherein A, C, G, T are respectively 489 (24.04%), 625 (30.73%), 520 (25.57%), 400 (19.67%).At the 253bp place initiator codon ATG is arranged, terminator codon TAA is arranged, the promoter sequence CGGCATTGCAATGCTTTCATCGAATGCTTTATTTTATTATGCAGGGGGAT of a supposition is arranged at 198bp at the 1450bp place.This sequence intronless has the complete opening code-reading frame of 1200bp, 399 amino acid of encoding.The coded product molecular weight is 43.22kD, and iso-electric point pI is 5.93.By GenBank gene order compare of analysis, show that Acetokinase gene Back of the present invention is a new Acetokinase gene, with registered E.C. 2.7.2.1 homology be 49～58%, wherein, prove a new bacterium Acetokinase gene with the Acetokinase gene homology the highest (58%) of Thermotoga maritima MSB8.This gene has expressive function, isolating Back gene is connected with prokaryotic expression carrier pet-22b, be built into the pet-22b-Back recombinant expression vector, (45～50kD), confirmation is a gene with expressive function to give expression to this gene protein after IPTG induces.The present invention is the separating acetic acid kinase gene from Ethanologenbacterium hit B49 genome, enlarge the Acetokinase gene resource, 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 a pMD18-T carrier structure collection of illustrative plates, and Fig. 3 is the restriction enzyme site structure iron of pMD18-T carrier, and Fig. 4 is TaKaRa LAPCR ^TMThe pcr amplification schematic diagram of in vitro cloning Kit test kit, Fig. 5 is Auele Specific Primer design attitude figure, and Fig. 6 is a pet-22b carrier structure collection of illustrative plates, and Fig. 7 is the restriction enzyme site structure iron of pet-22b carrier.

Embodiment

Embodiment one: the Ethanologenbacterium hit B49 Acetokinase gene Back described in the present embodiment is to be template with the total DNA of highly effective hydrogen yield bacterium Ethanologenbacterium hit B49, obtains by pcr amplification.Its concrete grammar is as follows:

1, the clone of Acetokinase gene:

(1) PCR primer design:

According to the bacterium E.C. 2.7.2.1 protein sequence of including among the GenBank (concrete bacterial classification sees Table 1), carry out the sequence alignment analysis with Blockmaker (http://blocks.fhere.org/blocks/blockmkr/make-blocks.html), then comparison result is imported the online primer-design software design of CODEHOP (http://blocks.fhere.org/blocks/codehop.html) primer, selected one couple of PCR amplification degenerated primer for use, concrete sequence sees Table 2:

The proteinic bacterial classification of the table 1 design used E.C. 2.7.2.1 of Acetokinase gene degenerated primer

Protein sequence	Bacterial classification	Number of registration
			E.C. 2.7.2.1	Clostridium thermocellum ATCC 27405	ZP_0031t922
E.C. 2.7.2.1	Clostridium acetobutylicum ATCC 824
			E.C. 2.7.2.1	Clostridium tetani E88
E.C. 2.7.2.1	Thermoanaerobacterium thermosaccharolyticum
			E.C. 2.7.2.1	Thermoanaerobacter tengcongensis MB4
E.C. 2.7.2.1	Clostridium perfringens str.13	NP_562640
			E.C. 2.7.2.1	Clostridium perfringens str.13	NP_561133

The degenerated primer of the amplification E.C. 2.7.2.1 of table 2.CODEHOP software design

The primer title	Primer sequence	Degeneracy
			ACKJl-forward (5 ' end primer)	5’TGAAAATGTATTAGCAAAAGGATTAGTAganmgnathgg3’	96
ACKJl-reverse (3 ' end primer)	5’CCCATTGCTAATCCTTCTAAAGGngtraancccat3’	32

Annotate: N (A, C, T or G), R (A or G), H (non-G), B (non-A), D (non-C).

(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 Acetokinase 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 ACKJ1-forward, ACKJ1-reverse, ExTaqDNA polysaccharase 2.5U, template DNA 0.2～1 μ g adds ddH ₂O to 50 μ L.Response procedures: pre-95 ℃ of 5min of sex change, 94 ℃ of 30s; 62 ℃ drop to 50 ℃, fall 1 ℃, 40s at every turn; 72 ℃ of 2min, totally 12 circulations are then by 94 ℃ of 30s; 50 ℃ of 40s; 72 ℃ of 2min, 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.PMDl8-T 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 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.Reclaim product and be connected linked system with the pMD18-T cloning vector: pMD18-T carrier 1 μ l (50ng), (100～200ng), Ligation Solution 5 μ l add 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.coliDH5a 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 lIPTG and 40 μ lX-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.

(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 Acetokinase gene part fragment both sides sequence C assette PCR:

Adopt TaKaRaLAPCR ^TMIn vitro cloning Kit test kit clone Acetokinase gene fragment both sides sequence.Based on the Acetokinase gene fragment sequence of having cloned, Cassette primerC1, Cassette primerC2 primer mate respectively in design and the test kit upstream primer ACK-Sll-lower, ACK-S12-lower and downstream primer ACK-S21-upper, ACK-S22-upper, concrete sequence sees Table 3.

The Cassette PCR primer of table 3 amplification Acetokinase gene part fragment both sides sequence

The primer title	Primer sequence
		Cassette primer C1	5’GTACATATTGTCGTTAGAACGCGTAATACGACTCA3’
Cassette primer C2	5’CGTTAGAACGCGTAATACGACTCACTATAGGGAGA3’
		ACK-S11-lower(C1)	5’CGAACGCGGTACTGCTCATAATATTTATTACGG3’
ACK-S12-lower(C2)	5’CGTCGATGCCTATCCGCTCTACTAATCCTTT3’
		ACK-S2l-upper(C1)	5’AAAAGCCGGAAGAAACCAAGATCGTC3’
ACK-S22-upper(C2)	5’GACACCAGCATGGGCTTTACACCTTTAGA3

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 (PrimerC2, PrimerS12 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 step of this test kit principle is poly-as follows:

1) with EcoR I restriction enzyme Ethanologenbacterium hit B49 genomic dna is decomposed fully.

2) carry out ligation with Cassette joint with EcoR 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 3 ends of primer are not wanted AT and are concentrated.

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 of 3 ends bases 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 Acetokinase gene part fragment both sides sequence:

According to the Acetokinase 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 LAPCR ^TMThe restriction enzyme that this enzyme joint is arranged in the in vitro cloning Kit test kit is selectedly at last cut genomic dna with EcoR I enzyme, the genomic dna after enzyme is cut be connected 30h with EcoR I adaptor joint in 16 ℃.Cassette PCR 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-dna solution 1.	34.5μl
		10×LA PCR Buffer II(Mg 2+plus)	5μl
TaKaRa LA Taq	0.5μl
		dNTP Mixture	8μl
Primer C1	1μl
		PrimerS11 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-1st PCR 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
		PrimerS12 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 E.C. 2.7.2.1 full-length gene, clone and order-checking:

According to the sequences Design total length checking primer ACK-upper and the ACK-lower that have spliced, be template amplification total length Acetokinase gene with Ethanologenbacterium hit B49 genome DNA.The PCR reaction system is 94 ℃ of 5min, 94 ℃ of 30s, and 58 ℃ of 30s, 72 ℃ of 1min20s, totally 35 circulations, last 72 ℃ are extended 10min, and the primer sees Table 8.

Table 8 Acetokinase gene total length checking primer

The primer title	Primer sequence
		ACK-upper	5’CGTCAAAACCGTCATAAAGCAAACAGGATCCC3’
ACK-lower	5’AGGAATAACCGGCAATACATAAAAGGCGATGGAA3’

(9) prokaryotic expression of Ethanologenbacterium hit B49 Acetokinase gene:

With Acetokinase 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.

According to the multiple clone site sequence of pet-22b expression vector, locate to introduce Nde I restriction enzyme site at the 253bp of Acetokinase 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, 55 ℃ of annealing 30s, 72 ℃ are extended 1min30s, totally 35 circulations; Last 72 ℃ are extended 10min.Glue reclaims the PCR product, cuts through Nde I and Xho I enzyme, reclaims at glue, is connected 3h with the pet-22b prokaryotic expression carrier that Nde 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 Acetokinase gene is expressed and is used primer

The primer title	Primer sequence
		ACK-22b-forward	5’ATGCAGGGGGATCAcatATGAAAATCTTGGTCATC3’
ACK-22b-reverse	5’GTTAATCTAGAGCCGACGAGTGTCTTGGTGTCGAGCGCGAT3’

(10) Ethanologenbacterium hit B49 E.C. 2.7.2.1 vitality test:

A, E.C. 2.7.2.1 vitality test:

Reaction solution: 145mM Tris-HCl, 200mM potassium acetate, the neutral NH2OH-HCl of 10mM ATP, 0.7M, 333 μ L.

Measure: the cell crude extract is added initial action in the above-mentioned reaction solution, and 25 ℃ of 20min add the trichoroacetic acid(TCA) termination reaction of final concentration 5% then.In reactant, add 2.5%FeCl ₂(being dissolved in 2N HCl) to 1mL in 25 ℃ of colour developings 15 minutes, centrifugal 5 minutes of 13000 * g removes insoluble substance, in A540nm place absorbancy.

B, protein content determination: adopt Shen, the Shanghai energy biotechnology BCA of company limited protein content determination test kit.

The definition (U) of C, enzyme unit alive: micromole's number of the ATP product of every milligram of (milligram) albumen per minute consumption.The E.C. 2.7.2.1 vigor is 70U.

Ethanologenbacterium hit B49 Acetokinase gene Back of the present invention is an E.C. 2.7.2.1, and registered sequence there are differences among this gene of Blast homology comparison result shows of sequence and the GenBank, is a new gene.

E.C. 2.7.2.1 Back gene sequence characteristic: this full length gene 2034bp, wherein A, C, G, T are respectively 489 (24.04%), 625 (30.73%), 520 (25.57%), 400 (19.67%).At the 253bp place initiator codon ATG is arranged, terminator codon TAA is arranged, the promoter sequence CGGCATTGCAATGCTTTCATCGAATGCTTTATTTTATTATGCAGGGGGAT of a supposition is arranged at 198bp at the 1450bp place.This sequence intronless has the complete opening code-reading frame of 1200bp, 399 amino acid of encoding.The coded product molecular weight is 43.22kD, and iso-electric point pI is 5.93.

Sequence table

One, E.C. 2.7.2.1 Back gene and coded product sequence thereof:

＜110〉Harbin Institute of Technology

＜120〉Acetokinase gene

<160>1

<210>1

<211>2034

<212>DNA

＜213〉E.C. 2.7.2.1 Back gene and coded product sequence thereof

<400>1

1 GCTCACACCGTTCCTTCCCGCCGTTTGCCGCGCGCCTGACCGCCCGCCGCAAACAACCAT

61 GCCCTGATGCGATTCTGATGCAGACGCAGCCGGCCCGCAGCCTGGCTCCTGTGCGTCAAA

121 ACCGTCATAAAGCAAACAGGATCCCGCATACTACTTGCAACTGCTTTCCCTACCTTATAT

181 AATAGTACATGTTGTATCGGCATTCATATGCTTTCATCGAATGCTTTATTTTATTATGCA

241 GGGGGATCACATATGAAAATCTTGGTCATCAACGCGGGAAGTTCTTCGCTGAAATACCAA

M K I L V I N A G S S S L K Y Q

301 CTGATCAACGTGGAGAACAAGGACGTGCTGGCAAAAGGCAACTGCGAGCGCATCGGCATC

L I N V E N K D V L A K G N C E R I G I

361 GACGGGCGGTTCAAGCACAAAACAGGCGACGGCCGCGGGTTTGAGCGCGAaGTGGCGCTC

D G R F K H K T G D G R G F E R E V A L

421 CCCGACCACCGCGCGGCGTTCCGTCTGGTTATCGAAGCCCTTACCACCGGTGAATTTGCC

P D H R A A F R L V I E A L T T G E F A

481 GTAATCGGCAGCACCAGTGAGATCGACGCGGTGGGCCACCGCATCGTGCACGGCGGCGAT

V I G S T S E I D A V G H R I V H G G D

541 AAGTTCACACACTCCGTGCTGGTGACCGACGAGGTGCTCAAAGAATTTCAGGGAGTCATC

K F T H S V L V T D E V L K E F Q G V I

601 AATTTCGCCCCCCTGCACAACCCTCCGGCACTCAGCGGCATTGAGGCCTGCCGGGAGACA

N F A P L H N P P A L S G I E A C R E T

661 CTGGGCAAAGCTGTGCCCAACGTCATGGTGTTCGACACCGCATTCCACCAGACCATGCCG

L G K A V P N V M V F D T A F H Q T M P

721 CCCCAGGCCTACATCTTCGGCGTGCCGTATAAATATTATGAGCAGTACCGCGTTCGCCGC

P Q A Y I F G V P Y K Y Y E Q Y R V R R

781 TACGGCGCGCATGGCACCTCCCACCGCTATGTCAGCCTGCGTTGCGCGGAGCTGCTGGGT

Y G A H G T S H R Y V S L R C A E L L G

841 AAAAAGCCGGAAGAAACCAAGATCGTCACCTGCCATCTGGGCAACGGTTCCTCCATCTCC

K K P E E T K I V T C H L G N G S S I S

901 GCGGTGGACGCCGGCAAATGTGTGGACACCAGCATGGGCTTTACACCTTTAGGCGGCATC

A V D A G K C V D T S M G F T P L G G I

961 ATCATGGGCACCCGCAGCGGCGACCTCGACCCCTCGGTGGTCACGCTTATCATGGAAAAA

I M G T R S G D L D P S V V T L I M E K

1021 GAAGGAATCAGCCCGCGCGATATGGAAAACCTGCTGAACAAGGAATCCGGCTTCATCGGC

E G I S P R D M E N L L N K E S G F I G

1081 ATCTCCGGTATCTCCAGCGACGACCGCGACCTGGAGGAAGCCACCGCCAAGGGCATTGAG

I S G I S S D D R D L E E A T A K G I E

1141 CGCTCGAAGATTGCGCAGGACGCCCAGCGCTATCAGATCAAAAAACTCGTCGGCGCTTAT

R S K I A Q D A Q R Y Q I K K L V G A Y

1201 TCGGCGGCCATGGGCGGCCTGGACGCGCTGGCGTTCACCGGCGGCATCGGCGAAAATTCC

S A A M G G L D A L A F T G G I G E N S

1261 TCGCTGCTGCGCGCAGCCGTCTGCGAAAACATGGAATACCTCGGCATCACCTTCGACCCC

S L L R A A V C E N M E Y L G I T F D P

1321 GCGAAAAACGAAGAAACCATCCGCGGCAAAGAGGGCGACCTTTCCCTGCCCGGCGGCGAA

A K N E E T I R G K E G D L S L P G G E

1381 GTGCGCGTTTATGTTATCCCCACCAACGAGGAATATATGATCGCGCTCGACACCAAGACA

V R V Y V I P T N E E Y M I A L D T K T

1441 CTCGTCGGCTAAAAGCTTCTAATAAAACGAATATATAAAAAGCGACGTTCCAAGATCTAG

L V G *

1501 AACGTCGCTTTTTAACATTCCGCCGCCGCGGAATGCGTTTTCCATACAGATGCGCGGCAA

1561 TGGCGGCACTGCTTGCTTTATGACGTGTGATGCGACTCCACAATCTCGCGTTCGCGAAAC

1621 AACAGCGAAATGCACCAGATGCCCGCGATGGCCACGAGCGTGAAGATGATACGGCTGACA

1681 GCGGAACCGGCTCCACCGAATGCCCAGTCGACCACATCAAAATTAAAAATGCCGACGCTG

1741 CCCCAGTTAAGCGCGCCAATAATCACCAGCAGCAAAGCGATCCTGTCCAGCATGAAATCA

1801 ACTCCCTTTTGCTTGCAGCCGCGCGGTTTCCTTTACGGTAACCGCAACGGTCCGTCAAAG

1861 GTTCAAAAAACAGATGAAGCATTCAGCTACCATCCGCTTTCGCAGATAGTATGGACAAAA

1921 ACGGGCGGATTATGCATCCGCCCGTTCCATCGCCTTTTATGTATTGCCGGTTATTCCTCG

1981 GCGCCGCTTCTCTGCTCTTTGAGCAAATCGCGGATCTCCTGCAAGACCACCAGA

Claims (1)

1, Acetokinase gene is characterized in that the gene order of described E.C. 2.7.2.1 is:

GCTCACACCGTTCCTTCCCGCCGTTTGCCGCGCGCCTGACCGCCCGCCG

CAAACAACCATGCCCTGATGCGATTCTGATGCAGACGCAGCCGGCCCGC

AGCCTGGCTCCTGTGCGTCAAAACCGTCATAAAGCAAACAGGATCCCGC

ATACTACTTGCAACTGCTTTCCCTACCTTATATAATAGTACATGTTGTATCG

GCATTCATATGCTTTCATCGAATGCTTTATTTTATTATGCAGGGGGATCACA

TATGAAAATCTTGGTCATCAACGCGGGAAGTTCTTCGCTGAAATACCAAC

TGATCAACGTGGAGAACAAGGACGTGCTGGCAAAAGGCAACTGCGAGC

GCATCGGCATCGACGGGCGGTTCAAGCACAAAACAGGCGACGGCCGCG

GGTTTTGAGCGCGAAGTGGCGCTCCCCGACCACCGCGCGGCGTTCCGTCT

GGTTATCGAAGCCCTTACCACCGGTGAATTTGCCGTAATCGGCAGCACCA

GTGAGATCGACGCGGTGGGCCACCGCATCGTGCACGGCGGCGATAAGTT

CACACACTCCGTGCTGGTGACCGACGAGGTGCTCAAAGAATTTCAGGGA

GTCATCAATTTCGCCCCCCTGCACAACCCTCCGGCACTCAGCGGCATTGA

GGCCTGCCGGGAGACACTGGGCAAAGCTGTGCCCAACGTCATGGTGTTC

GACACCGCATTCCACCAGACCATGCCGCCCCAGGCCTACATCTTCGGCGT

GCCGTATAAATATTATGAGCAGTACCGCGTTCGCCGCTACGGCGCGCATG

GCACCTCCCACCGCTATGTCAGCCTGCGTTGCGCGGAGCTGCTGGGTAA

AAAGCCGGAAGAAACCAAGATCGTCACCTGCCATCTGGGCAACGGTTCC

TCCATCTCCGCGGTGGACGCCGGCAAATGTGTGGACACCAGCATGGGCT

TTACACCTTTAGGCGGCATCATCATGGGCACCCGCAGCGGCGACCTCGAC

CCCTCGGTGGTCACGCTTATCATGGAAAAAGAAGGAATCAGCCCGCGCG

ATATGGAAAACCTGCTGAACAAGGAATCCGGCTTCATCGGCATCTCCGGT

ATCTCCAGCGACGACCGCGACCTGGAGGAAGCCACCGCCAAGGGCATT

GAGCGCTCGAAGATTGCGCAGGACGCCCAGCGCTATCAGATCAAAAAAC

TCGTCGGCGCTTATTCGGCGGCCATGGGCGGCCTGGACGCGCTGGCGTT

CACCGGCGGCATCGGCGAAAATTCCTCGCTGCTGCGCGCAGCCGTCTGC

GAAAACATGGAATACCTCGGCATCACCTTCGACCCCGCGAAAAACGAAG

AAACCATCCGCGGCAAAGAGGGCGACCTTTCCCTGCCCGGCGGCGAAG

TGCGCGTTTATGTTATCCCCACCAACGAGGAATATATGATCGCGCTCGACA

CCAAGACACTCGTCGGCTAAAAGCTTCTAATAAAACGAATATATAAAAAG

CGACGTTCCAAGATCTAGAACGTCGCTTTTTAACATTCCGCCGCCGCGGA

ATGCGTTTTCCATACAGATGCGCGGCAATGGCGGCACTGCTTGCTTTATG

ACGTGTGATGCGACTCCACAATCTCGCGTTCGCGAAACAACAGCGAAAT

GCACCAGATGCCCGCGATGGCCACGAGCGTGAAGATGATACGGCTGACA

GCGGAACCGGCTCCACCGAATGCCCAGTCGACCACATCAAAATTAAAAA

TGCCGACGCTGCCCCAGTTAAGCGCGCCAATAATCACCAGCAGCAAAGC

GATCCTGTCCAGCATGAAATCAACTCCCTTTTGCTTGCAGCCGCGCGGTT

TCCTTTACGGTAACCGCAACGGTCCGTCAAAGGTTCAAAAAACAGATGA

AGCATTCAGCTACCATCCGCTTTCGCAGATAGTATGGACAAAAACGGGCG

GATTATGCATCCGCCCGTTCCATCGCCTTTTATGTATTGCCGGTTATTCCTC

GGCGCCGCTTCTCTGCTCTTTGAGCAAATCGCGGATCTCCTGCAAGACCA

CCAGA。

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