CN103087998B - Enzyme for synthesizing cetyl-coenzyme A through cordyceps sinensis, gene and application thereof - Google Patents
Enzyme for synthesizing cetyl-coenzyme A through cordyceps sinensis, gene and application thereof Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/1025—Acyltransferases (2.3)
- C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/26—Preparation of nitrogen-containing carbohydrates
- C12P19/28—N-glycosides
- C12P19/30—Nucleotides
- C12P19/32—Nucleotides having a condensed ring system containing a six-membered ring having two N-atoms in the same ring, e.g. purine nucleotides, nicotineamide-adenine dinucleotide
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- C12Y203/00—Acyltransferases (2.3)
- C12Y203/01—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
- C12Y203/01086—Fatty-acyl-CoA synthase (2.3.1.86)
Abstract
The invention relates to long-chain-acyl-coenzyme A synthetase for synthesizing metabolic cetyl-coenzyme A with the start that Bailing producing strain cordyceps sinensis hirsutella sinensis is participated in palmatic acid, a gene for coding the enzyme and an application thereof. The amino acid sequence of the long-chain-acyl-coenzyme A synthetase and the sequence represented by SEQ ID No. 1 or SEQ ID No. 3 have more than 90% of homology. According to the invention, the metabolic pathway for synthesizing cetyl-coenzyme A through palmatic acid is researched in principle in a detailed manner as follows: clone DNA of a nucleotide sequence provided by the invention can be transferred into engineering bacteria through transduction, conversion and combined transfer methods; gene expression is biologically synthesized by adjusting the cetyl-coenzyme A; high expressivity of host long-chain-acyl-coenzyme A synthetase is given; an effective way for enlarging the yield of the cetyl-coenzyme A is provided; and the cetyl-coenzyme A has a significant application prospect.
Description
(1) technical field
The present invention relates to set out long-chain-acyl group-coenzyme A synthetic enzyme (long-chain acyl-CoA synthetase) of anabolism hexadecyl-coenzyme A of a kind of participation palm fibre eleostearic acid of producing bacterium Cordyceps sinensis China pilose spore from " hundred make ", the gene of this enzyme of encoding and application thereof.
(2) background technology
Cordyceps sinensis (Cordyceps sinensis (Berk.) Sacc.) is that Cordyceps fungus colonizes in stroma and the complex body on larva corpse (comprising stroma and polypide) on lepidopteran (Lepidoptera) Hepialidae insect (Hepialus armoricanus Oberthur) larva.Cordyceps sinensis is traditional fungi herb resource that a class is treasured, and has the various feature of meta-bolites and biological activity, at biomedicine field, shows huge application and development prospect.Cordyceps sinensis with its multiple medicinal efficacy extensively, obviously receive much concern, worldwide enjoys high praise.The traditional Chinese medical science thinks, Cordyceps sinensis enters lung kidney two warps, can tonifying lung cloudy, again can kidney-replenishing, and cure mainly and suffer from a deficiency of the kidney, impotence and seminal emission, soreness of waist and knee joint, weak after being ill, chronic cough is weak, phthisical cough phlegm blood, spontaneous sweatings etc., are unique a kind of balance simultaneously, the Chinese medicine that regulates negative and positive.Modern pharmacology confirms, and Cordyceps sinensis has the biological activity widely such as immunomodulatory, antibacterial, antitumor, anti-oxidant, anti-ageing, hypoglycemic blood fat, gonadotropic Effect.
Cordyceps fungus is a kind of ascomycetes, has Conidial Stage (anamorph) and thecaspore stage (teleomorph) in its life history.And what use in the actual productions such as artificial culture, liquid fermenting is the Cordyceps fungus in imperfect stage, thereby the evaluation of Anamorph of Cordyceps Sinensis is extremely important.Chinese scholars is being done a lot of work aspect Cordyceps Resources investigation, anamorph confirmation, activeconstituents compartment analysis and the mechanism of action, Application and Development.Cordyceps sinensis China pilose spore has been proved to be the anamorph existence form of Cordyceps sinensis, has the activeconstituents identical with natural cordyceps and drug effect.
Natural cs has strict parasitics and special ecotope, therefore its output is very low, price is high.Wild cordyceps is because factors such as being subject to growing environment restricts, scarcity of resources.Owing to making little progress on artificial culture in recent years, the research of wild cordyceps surrogate focuses mostly on liquid fermenting.Utilizing liquid submerged fermentation to cultivate Cordyceps mycelium, extract or fermented liquid, is a kind of effective way that solves Cordyceps sinensis medicine source.Chinese caterpillar fungus fermentation is produced Chinese caterpillar fungus substitute, both can effectively protect these precious resources of Chinese caterpillar fungus, and not climate, geographical environment and the strict restriction of Chinese caterpillar fungus parasitic conditions is again suitable for large-scale industrialization and produces.The substitute of producing is as also similar to natural cs with drug effect in its composition of mycelium, thereby is devoted to the fermentation culture of Cordyceps mycelium both at home and abroad always.The mycelia that aweto cultured by artificial fermentation China pilose spore obtains, through toxicity, pharmacology, plant research, proof is basically identical with natural cs chemical constitution, pharmacological action, can replace natural cs to produce cordyceps product, to make up the shortage of natural resources, by the optimization to fermentation condition, the amount of mycelial biomass and meta-bolites is all significantly improved.
In recent years, along with the develop rapidly of natural product chemistry and modern chromatographic technique, to progressively turning to deeper functional meta-bolites to study by the direct utilization of Chinese caterpillar fungus raw material or crude extract in worm grass product research and development.Chinese caterpillar fungus meta-bolites has been done to a large amount of research both at home and abroad, meta-bolites mainly comprises several large compounds such as nucleosides, polysaccharide, polypeptide, sterol, and wherein the research of the representative functional meta-bolites such as purines nucleosides, Cordyceps polysaccharide, N.F,USP MANNITOL at aspects such as biosynthesizing, pharmacological actions wins initial success.
Unsaturated fatty acids refers to the lipid acid that contains one or more pairs of keys in molecule, and its fusing point is low compared with saturated fatty acid.Unsaturated fatty acids is a kind of lipid acid that forms body fat, the lipid acid of needed by human, and unsaturated fatty acids, according to the difference of two key numbers, is divided into two kinds of monounsaturated fatty acids and polyunsaturated fatty acids.Polyunsaturated fatty acid (Polyunsaturated Fatty Acids, PUFAs) relative saturation lipid acid has more effect, it can reduce blood cholesterol and triglyceride level, regulate heart function, reduce blood viscosity, improve blood microcirculation, improve the activity of brain cell, memory and thinking ability, strengthen human defensive system's function etc., in addition it can also get rid of unnecessary " rubbish " in human body, namely due to the superabundant fat that forms of excessive saturated fatty acid of having taken the photograph people, thereby reaches the object of fat-reducing.Therefore, its potential medical pharmaceutical use has been subject to the extensive concern in the world, has caused the great attention of the industries such as food, medical even makeup.
Yung-Sheng in 1999 draws and has cloned △ 6 and △ 12 fatty acid dehydrogenase genes of Mortierella alpina (Mortiere Uaalpina) and expressed in yeast saccharomyces cerevisiae.2004, the people such as Dyer proceeded to yeast by 3 desaturases of the △ in tung oil tree, had successfully obtained and had produced linolenic yeast.The people such as Maali-Amiri in 2007 proceed to potato by △ 12 desaturases of the blue or green bacterium of algae (Cyanobacterium), potato fatty acid component successfully detected considerable change has occurred.2008, the people such as Hao turned △ 6 desaturases of volume branch Mucor in people's transgene tobacco, have successfully obtained the bacterial strain of high yield gamma-linolenic acid.In addition, also have the gene that many fatty acid desaturases are relevant to be cloned and Transformation Application.Because most of desaturase is embrane-associated protein, its separation and purification is very difficult, and separation and purification the desaturase of identifying are very few, and most research is carried out around delta 8 desaturase genes and expression regulation thereof.
At present, applied unsaturated fatty acids production bacterium be take subtilis as main, and as the Cordyceps fungus of important anabolism unsaturated fatty acids, also only rest in the research of meta-bolites composition analysis and effect, also rarely found to the research of genes involved and albumen in Cordyceps fungus unsaturated fatty acids metabolic pathway of synthesizing.
(3) summary of the invention
The object of the invention is for the deficiency of above existence and the technical issues that need to address; " hundred make " produced to long-chain-acyl group-coenzyme A synthetic enzyme and the encoding gene thereof of bacterium Cordyceps sinensis China pilose spore anabolism hexadecyl-coenzyme A and further investigate, provide " hundred makes " production bacterium Cordyceps sinensis China pilose spore to participate in brown eleostearic acid set out long-chain-acyl group-coenzyme A synthetic enzyme, encoding gene and the application thereof of anabolism hexadecyl-coenzyme A.
The technical solution used in the present invention is:
Participate in set out long-chain-acyl group-coenzyme A synthetic enzyme of anabolism hexadecyl-coenzyme A of brown eleostearic acid, shown in this enzyme and SEQ ID No.1 or SEQ ID No.3, sequence has 90% above homology.This enzyme can be prepared corresponding hexadecyl-coenzyme A by catalysis palm fibre eleostearic acid.Singularity due to aminoacid sequence; any fragment or its variant that contains the peptide protein of aminoacid sequence shown in SEQ ID NO.1 or SEQ ID No.3; as its examples of conservative variations, bioactive fragment or derivative; as long as the fragment of this peptide protein or peptide protein variant and aforementioned amino acid sequence homology, more than 90%, all belong to the row of protection domain of the present invention.Concrete described change can comprise amino acid whose disappearance, insertion or replacement in aminoacid sequence; Wherein, for the conservative property change of variant, the amino acid of replacing has the structure similar to original acid or chemical property, and as replaced Isoleucine with leucine, variant also can have non-conservation and change, as replaced glycine with tryptophane.
Preferably, described long-chain-acyl group-coenzyme A synthetic enzyme aminoacid sequence (is designated as respectively unsC as shown in SEQ ID No.1 or SEQ ID No.3
1, unsC
2albumen).
Long-chain-acyl group of the present invention-coenzyme A synthetic enzyme is produced bacterium Cordyceps sinensis China pilose spore from " hundred make ".
The path that obtains corresponding hexadecyl-coenzyme A by each brown eleostearic acid anabolism is as follows:
The invention still further relates to described long-chain-acyl group-coenzyme A synthetic enzyme and prepare the application in hexadecyl-coenzyme A at biocatalysis palm fibre eleostearic acid.
The invention still further relates to the encoding gene of above-mentioned long-chain-acyl group-coenzyme A synthetic enzyme, i.e. long-chain-acyl group-coenzyme A synthase gene.Concrete, this encoding gene can be the gene order with polynucleotide shown in SEQ ID NO:2 or SEQ ID No.4 with 90% above homology.Due to the singularity of nucleotide sequence, the variant of polynucleotide shown in any SEQ ID NO:2 or SEQ ID No.4, as long as itself and this polynucleotide have 90% above homology, all belongs to the row of protection domain of the present invention.The variant of described polynucleotide refers to a kind of polynucleotide sequence that one or more Nucleotide changes that has.The variant of these polynucleotide can make raw displacement varient or the varient of non-life, comprises and replaces varient, deletion mutation body and insert varient.As known in the art, allelic variant is the replacement form of polynucleotide, and it may be replacement, disappearance or the insertion of polynucleotide, but can be from not changing in fact the function of the peptide protein of its coding.
Preferably, the nucleotide sequence of described gene (is designated as unsC as shown in SEQ ID No.2 or SEQ ID No.4
1, unsC
2gene, unsC
1genes encoding unsC
1albumen, unsC
2genes encoding unsC
2albumen).
Described gene can be used for building the genetic engineering bacterium of can biocatalysis palm fibre eleostearic acid preparing hexadecyl-coenzyme A, to expand the output of hexadecyl-coenzyme A or derivatives thereof.
Main points of the present invention have been to provide the nucleotide sequence shown in the aminoacid sequence shown in SEQ ID NO:1 or 3 and SEQ ID NO:2 or 4, the in the situation that of known this aminoacid sequence and nucleotide sequence, the acquisition of this aminoacid sequence and nucleotide sequence, and the acquisition of related vector, host cell, be all apparent to those skilled in the art.
Beneficial effect of the present invention is mainly reflected in: the present invention studies in detail the synthetic hexadecyl-coenzyme A pathways metabolism of brown eleostearic acid principle; the cloned DNA that comprises nucleotide sequence provided by the present invention can be used for by transduction, transform, in conjunction with the method shifting, proceeds in engineering bacteria; by regulating the expression of hexadecyl-coenzyme A biosynthesis gene; give the high expression level of host's long-chain-acyl group-coenzyme A synthetic enzyme; for expanding the output of hexadecyl-coenzyme A or derivatives thereof, provide effective way, there is major application prospect.
(4) accompanying drawing explanation
Fig. 1 is the denaturing formaldehyde gel electrophoresis figure that " hundred make " produces the total RNA of bacterium Cordyceps sinensis China pilose spore;
Fig. 2 is Fatty acid biosynthesis metabolism approach annotated map;
Fig. 3 is fatty acid metabolism approach annotated map;
Fig. 4 is unsaturated fatty acids metabolic pathway of synthesizing annotated map;
Fig. 5 is long-chain-acyl group-coenzyme A synthase gene pcr amplification product gel electrophoresis figure;
Fig. 6 is cloning vector pMD18-T Vector and expression vector pET-28a physical map;
Fig. 7 is restructuring cloned plasmids pMD18-T/uns C physical map;
Fig. 8 is recombinant expression plasmid pET-28a/uns C building process schematic diagram;
Fig. 9 is recombinant expression plasmid pET-28a/uns C physical map;
Figure 10 is the SDS-PAGE figure of long-chain-acyl group-coenzyme A synthetase albumen.
(5) embodiment
Below in conjunction with specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in this:
Embodiment 1: " hundred make " produces the cultivation of bacterium Cordyceps sinensis China pilose spore
Bacterium source: first gather natural cordyceps from Qinghai, and taken back Hangzhou and carried out separation screening, obtained L0106 bacterial strain, and be China pilose spore (Hirsutella Sinensis) through this bacterial strain of strain identification, this culture presevation is at Chinese Typical Representative culture collection center, deposit number is CCTCC No:M 2011278, in the patent CN102373190A of previously application, discloses.
This bacterial classification is inoculated in to inclined-plane, (this is the liquid formulations before solidifying to culture medium prescription, in following ratio, prepare afterwards bevel again) be glucose 2.0%(w/v, 1% represents to contain 1g in 100mL substratum, Semen Maydis powder 1.0%, murphy juice 0.5%, dextrin 0.5%, yeast powder 0.5%, wheat bran 1.0%, dried silkworm chrysalis meal 2.0%, peptone 1.0%, magnesium sulfate 0.05%, potassium primary phosphate 0.05%, agar powder 1.0% down together),, surplus is water, at 12 ~ 16 ℃, cultivates 25 days; Then bacterial classification is inoculated in to fermention medium, culture medium prescription is glucose 1.0%, molasses 1.0%, dried silkworm chrysalis meal 0.5%, soybean cake powder 1.0%, yeast extract paste 0.5%, magnesium sulfate 0.01%, potassium primary phosphate 0.02%, surplus is water, be placed on shaking table, 12 ~ 16 ℃ of cultivations of temperature 25 days, under aseptic condition, carry out solid-liquid separation after cultivation finishes, and solid is placed in to aseptic utensil, standby.
Embodiment 2: " hundred make " produces the extraction of the total RNA of bacterium Cordyceps sinensis China pilose spore
With TRIzol reagent, extract total RNA, step is specially: 1) liquid nitrogen grinding: get the new fresh thalli of 1g and put into mortar, repeatedly add liquid nitrogen to be fully ground to Powdered, divide and install in the 1.5mL centrifuge tube of precooling, add 1mL TRIzol reagent, mix, standing 5min, makes nucleic acid-protein mixture completely separated on ice.2) RNA is separated: add 0.2mL chloroform, firmly concussion mixes 15s, standing 2 ~ 3min on ice, and 4 ℃, the centrifugal 15min of 12000rpm, layering, gets upper strata water, approximately 600 μ L.3) RNA precipitation: add 500 μ L Virahols, at standing 10min on ice, 4 ℃, the centrifugal 10min of 12000rpm, abandon supernatant.4) RNA washing: add 1mL 75%(v/v) ethanol, will precipitate and hang, standing 10min on ice, 4 ℃, the centrifugal 15min of 7500rpm; Repeat washing step above, then wash one time.5) dissolve RNA: centrifuge tube is placed in and opens wide dry 5 ~ 10min on ice, add appropriate DEPC water dissolution.
Embodiment 3: " hundred make " produces the order-checking of bacterium Cordyceps sinensis China pilose spore RNA sample
Extract after the total RNA of sample, use with Oligo(dT) enrichment with magnetic bead mRNA.Add fragmentation buffer that mRNA is broken into short-movie section (200 ~ 700bp), take mRNA as template, with the synthetic article one cDNA chain of hexabasic base random primer (random hexamers), then synthesize second cDNA chain, through QiaQuick PCR test kit purifying and after adding EB buffer solution elution, do end reparation, add polyA and connect sequence measuring joints again, then with agarose gel electrophoresis, carry out clip size selection, finally carry out pcr amplification, the sequencing library of building up checks order with Illumina GA IIx.The raw image data that order-checking obtains is converted into sequence data through base calling, i.e. raw data or raw reads.Remove the reads that only contains adaptor sequence in primitive sequencer reads, standby with subsequent analysis.
Embodiment 4: " hundred make " produces the short sequence assembling of reading of bacterium Cordyceps sinensis China pilose spore RNA
Use short reads composite software SOAPdenovo(Li, Zhu et al. De novo assembly of human genomes with massively parallel short read sequencing [J]. Genome Res, 2010,20:265-272.) transcribe group and from the beginning assemble.First SOAPdenovo is linked to be the reads with certain length overlap longer not containing the Contig fragment of N.Then reads is compared back to Contig, by paired-end reads, determine from the different Contig of same transcript and the distance between these Contig, SOAPdenovo connects together these Contig, and middle unknown nucleotide sequence represents with N, so just obtains Scaffold.Further utilize paired-end reads to do filling-up hole to Scaffold and process, finally obtain containing N minimum, the Unigene sequence that two ends can not extend again.Finally, Unigene sequence and albumen database nr, Swiss-Prot, KEGG and COG are done to blastx and compare (evalue<0.00001), get the sequence direction that the best albumen of comparison result is determined Unigene.If the comparison result between different sink is contradictory, press nr, Swiss-Prot, the priority of KEGG and COG is determined the sequence direction of Unigene, with above four storehouses all to less than Unigene software ESTScan(Iseli, Jongeneel et al. ESTScan:a program for detecting, evaluating, and reconstructing potential coding regions in EST sequences[J]. In Proceedings of 9th InternationalConference on Intelligent Systems for Molecular Biology. AAAIPress, Menlo Park, CA, pp. 1999, 138-148.) predict the direction of its coding region definite sequence.For the Unigene that can determine sequence direction, provide its sequence from 5' to 3' direction, for the Unigene that cannot determine sequence direction, provide the sequence that composite software obtains.
Embodiment 5: " hundred make " produces bacterium Cordyceps sinensis China pilose spore Unigene functional annotation
Functional annotation information provides protein function annotation, Pathway annotation, COG functional annotation and the Gene Ontology(GO of Unigene) functional annotation.First, by blastx by Unigene sequence alignment to albumen database nr, Swiss-Prot, KEGG and COG(evalue<0.00001), obtain thering is the albumen of highest serial similarity with given Unigene, thereby obtain the protein function annotation information of this Unigene.According to KEGG annotation information, can further obtain the Pathway annotation of Unigene.Unigene and COG database are compared, and the function that prediction Unigene is possible is also done function statistic of classification to it.According to nr annotation information, use Blast2GO software (Conesa, Gotz et al. Blast2GO:a universal tool for annotation, visualization and analysis in functional genomics research[J]. Bioinformatics, 2005,21 (18): the GO annotation information that 3674-3676.) obtains Unigene.Obtain after the GO annotation of each Unigene, with WEGO software (Ye, Fang et al. WEGO:a web tool for plotting GO annotations[J]. Nucleic Acids Research, 2006,34:293-297.) all Unigene are done to GO functional classification statistics, from macroscopic view, be familiar with the gene function distribution characteristics of these species.
Embodiment 6: " hundred make " produced bacterium Cordyceps sinensis China pilose spore hexadecyl-coenzyme A pathways metabolism and analyzed
Fig. 2 is the Fatty acid biosynthesis metabolism (map00061) in KEGG pathways metabolism annotation; Fig. 3 is the fatty acid metabolism (map00071) in KEGG pathways metabolism annotation; Fig. 4 is the unsaturated fatty acids anabolism (map01040) in KEGG pathways metabolism annotation; the enzyme having annotated is that " hundred make " that detected produces bacterium Cordyceps sinensis China pilose spore hexadecyl-coenzyme A pathways metabolism relevant enzymes; as can be seen from the figure, detected from 2 Unigene of long-chain-acyl group-coenzyme A synthetic enzyme of the synthetic corresponding hexadecyl-coenzyme A of brown eleostearic acid.By the ORF Finder software in NCBI, detect online, found out the open reading frame (SEQ ID No.2, SEQ ID No.4) of this gene and obtained corresponding protein sequence (SEQ ID No.1, SEQ ID No.3).
Embodiment 7: " hundred make " produces bacterium Cordyceps sinensis China pilose spore long-chain-acyl group-coenzyme A synthase gene design of primers
Each gene open reading frame DNA sequence dna design primer that uses GENE RUNNER primer-design software to obtain according to prediction; for clone's " hundred make ", produce long-chain-acyl group-coenzyme A synthase gene of bacterium China pilose spore anabolism hexadecyl-coenzyme A; primer is synthetic by Shanghai Sheng Gong biotechnology company limited, and primer sequence is listed as follows:
UnsC
1gene: forward primer 5 ' ATAGAATTCATGGACTCCTGGGGTCTGGAC3 '
Reverse primer 5 ' AGAAAGCTTTCACTCAGTGCCAGCATCGTC3 '
UnsC
1mrna length is 1239bp.
UnsC
2gene: forward primer 5 ' AGCGAATTCATGCCCCTCATGACAGGATAC3 '
Reverse primer 5 ' ATAAAGCTTTCACATGCCAGACAGGCCGC3 '
UnsC
2mrna length is 411bp.
Embodiment 8: " hundred make " produces the preparation of bacterium Cordyceps sinensis China pilose spore cDNA the first chain
The method first providing according to embodiment 1 is turned out after sutella sinensis fermented mycelium, the method providing according to embodiment 2 is again carried out the extraction of total RNA to China pilose spore, obtain by following, being undertaken synthesizing of " hundred make " production bacterium Cordyceps sinensis China pilose spore cDNA the first chain after total RNA, for follow-up each gene clone experiment.
Adopt synthetic cDNA the first chain of PrimeScript 1st Strand cDNA Synthesis Kit test kit (TaKaRa) reverse transcription from Total RNA, experimental procedure is as follows:
1) in Microtube pipe, prepare following mixed solution.
2) sex change, annealing operation are conducive to the sex change of template ribonucleic acid and the annealing of the specificity of reverse transcription primer and template, can improve reverse transcription reaction efficiency, so carry out sex change, annealing reaction on PCR instrument, condition setting is as follows:
65℃，5 min
3) annealing finishes the mixed solution that the rear centrifugal several seconds makes template ribonucleic acid/primer etc. and is gathered in Microtube pipe bottom.
4) in above-mentioned Microtube pipe, prepare following inverse transcription reaction liquid.
5) on PCR instrument, by following condition, carry out reverse transcription reaction.
42℃ 15～30 min
70℃ 15 min
Generalized case, at eukaryote mRNA 3 ' end, there is a PolyA structure, the quantity of A base ten to hundreds of not etc., utilize this structure can utilize Oligo (dT) primer, under the effect of ThermoScript II, take mRNA as synthetic cDNA the first chain of template, the sequence (providing in PrimeScript 1st Strand cDNA Synthesis Kit) in the dT region of being developed alone by TaKaRa is provided in the present invention is primer, if the mRNA integrity obtaining is better, by reverse transcription process, can obtain so cDNA first chain of all zymoprotein encoding genes in species.
Embodiment 9: " hundred make " produces the detection of clone, expression and the protein vigor of bacterium Cordyceps sinensis China pilose spore anabolism hexadecyl-coenzyme A functional gene long-chain-acyl group-coenzyme A synthetic enzyme unsC gene
1, the pcr amplification of long-chain-acyl group-coenzyme A synthetic enzyme unsC gene
CDNA the first chain obtaining in embodiment 8 of take is template, with synthetic unsC in embodiment 7
1gene primer: 5 ' ATA GAA TTC ATG GAC TCC TGG GGT CTG GAC3 ' and 5 ' AGA AAG CTT TCA CTC AGT GCC AGC ATC GTC3 ', unsC
2gene primer: 5 ' AGC GAA TTC ATG CCC CTC ATG ACA GGA TAC3 ' and 5 ' ATA AAG CTT TCA CAT GCC AGA CAG GCC GC3 ' carry out Pfu archaeal dna polymerase pcr amplification reaction, and condition setting is as follows:
Pfu pcr amplification reaction system:
Pfu DNA Ploymerase pcr amplification condition:
2, long-chain-acyl group-coenzyme A synthetic enzyme unsC gene PCR product gel electrophoresis detection
Concrete detection method is: 1) with microwave-oven-heating, it is uniformly dissolved 0.9% the sepharose preparing; 2) get 15mL gel, when gel is cooled to 50 ℃ of left and right, add 1 μ L staining fluid Gold view, after mixing, pour on treatments of Electrophoretic Slab Gels, remove and insert point sample comb after bubble; 3) after gel solidifies, carefully take out point sample comb, offset plate is put into electrophoresis chamber (one end, point sample hole is near the negative pole of electrophoresis chamber), in electrophoresis chamber, add TAE electrophoretic buffer; 4) get 5 μ L samples and then add 6 * Loading Buffer, 1.5 μ L and ddH
2after O 4 μ L mix, using liquid-transfering gun loading, applied sample amount is 10 μ L; 5) connect the supply lead between electrophoresis chamber and electrophoresis apparatus, just very red, negative pole is black; 6) power-on, starts electrophoresis, and maximum voltage is no more than 5 V/cm; 7) when sample ran offset plate 2/3 time can stop electrophoresis; 8), after cutting off the electricity supply, gel taken out and put into the observation of gel imaging instrument, take pictures.
Transcribe group order-checking prediction long-chain-acyl group-coenzyme A synthetic enzyme unsC
1the size of gene is 1239bp, unsC
2the size of gene is 411bp, and agarose gel electrophoresis result shows successfully to have amplified long-chain-acyl group-coenzyme A synthetic enzyme unsC
1the size of gene is about 1200bp, unsC
2the size of gene is about 400bp.Fig. 5 is that " hundred make " produces bacterium China pilose spore anabolism hexadecyl-coenzyme A functional gene PCR product gel electrophorogram.
3, the base A that adds of long-chain-acyl group-coenzyme A synthetic enzyme unsC gene PCR product processes and purifying
Because Pfu archaeal dna polymerase PCR product end is flush end, so just can be used for the connection of T carrier also need to add base A processing, purifying after glue reclaims after.It is as follows that glue recovery product adds base A system:
In PCR instrument, 72 ℃ add A base 20 min, finally with AxyPrep PCR cleaning agents box, purify.
4, long-chain-acyl group-coenzyme A synthetic enzyme unsC gene and cloning vector is connected
Cloning vector pMD18-T Vector is purchased from TaKaRa company (TaKaRa code D101A), and its physical map is shown in Fig. 6, by long-chain-acyl group-coenzyme A synthetic enzyme unsC
1, unsC
2gene is connected construction recombination plasmid pMD18-T/unsC with cloning vector
1, pMD18-T/unsC
2, physical map is shown in Fig. 7, linked system and condition of contact are as follows.
Linked system:
Condition of contact: 16 ℃, 16h; Deactivation: 65 ℃, 15min.
5, the conversion of long-chain-acyl group-coenzyme A synthetic enzyme recombinant plasmid pMD18-T/unsC
Recombinant plasmid pMD18-T/unsC is proceeded in intestinal bacteria E. coli JM109 and to build the recombinant bacterium E. coli JM109/pMD18-T/unsC that carries long-chain-acyl group-coenzyme A synthetic enzyme unsC gene, concrete steps are: 1) 10 μ L reaction systems are gone in competent cell E. coli JM109 to ice bath 30min; 2) thermal shock: 42 ℃, 90s; 3) ice bath: 2-3min; 4) add 800 μ L liquid LB, 37 ℃, 250rpm, 1h; 5) spread plate (containing Amp resistance); 6) 37 ℃ of incubator overnight incubation.
6, the screening of the positive recombinant bacterium of long-chain-acyl group-coenzyme A synthetic enzyme E. coli JM109/pMD18-T/unsC
Bacterium colony PCR can extract genomic dna, and directly take the DNA that exposes after thalline pyrolysis, carry out pcr amplification as template, the method is easy and simple to handle, quick, can Rapid identification bacterium colony whether be the positive bacterium colony that contains object plasmid, transform in identifying comparatively common.In experiment, by being inoculated into single bacterium colony corresponding in liquid nutrient medium, carry out bacterium colony PCR, to verify whether proceed to goal gene.First, with toothpick picking list bacterium colony, add containing in the 1.5mL centrifuge tube of 50 μ L sterilized waters, boiling water bath 30min, then centrifugally usings supernatant as template, carries out pcr amplification, and PCR program setting is Taq enzymatic amplification general procedure.Finally adopt 0.9% agarose gel electrophoresis detection bacterium colony PCR product.
7, the order-checking of long-chain-acyl group-coenzyme A synthetic enzyme recombinant plasmid pMD18-T/unsC
After the detected positive recombinant bacterium liquid LB culture medium culturing of bacterium colony PCR is spent the night, get 4mL bacterium liquid and extract plasmid, the operation instructions that method provides by AxyPrep plasmid DNA small volume of reagent box.Order-checking is completed by Sani bio tech ltd, Shanghai.Through sequence verification, sequence SEQ ID No.2 and SEQ ID No.4 are to recombinate to pMD18-T/unsC respectively
1and pMD18-T/unsC
2in.
8, the structure of long-chain-acyl group-coenzyme A synthetic enzyme recombinant expression plasmid pET-28a/unsC
Experimental basis foreign gene is in the principle of expression in escherichia coli, and expression vector pET-28a and long-chain-acyl group-coenzyme A synthetic enzyme unsC gene restriction enzyme site comparison situation, has determined unsC
1, unsC
2for EcoR I and Hind III double enzyme site, and recombination bacillus coli E. coli JM109/pMD18-T/unsC is carried out to the cultivation of liquid LB test tube shaker, recombinant plasmid extraction.
The recombinant plasmid pMD18-T/unsC of long-chain-acyl group-coenzyme A synthetic enzyme unsC gene and expression vector pET-28a use respectively EcoR I/Hind III restriction enzyme 37 ℃ respectively enzyme cut and process 6h, it is as follows that enzyme is cut system:
EcoR I/Hind III double digestion system:
Enzyme is cut and is finished rear 65 ℃ of deactivation 15min, then respectively with Axygen DNA gel recovery test kit reclaim, purifying.
Long-chain-acyl group-coenzyme A synthetic enzyme unsC gene and expression vector pET-28a spend the night with 16 ℃ of connections of T4 ligase enzyme after double digestion, purifying again; build recombinant expression plasmid pET-28a/unsC; its building process is shown in Fig. 8, builds the recombinant expression plasmid pET-28a/unsC obtaining
1, pET-28a/unsC
2fig. 9 is shown in by collection of illustrative plates.Linked system is composed as follows:
Linked system:
9, the conversion of long-chain-acyl group-coenzyme A synthetic enzyme recombinant expression plasmid pET-28a/unsC and the screening of positive monoclonal
The expression plasmid heat shock building is converted in E. coli BL21 Host Strains, is then applied on the LB agar plate that contains kantlex (Kan) resistance 37 ℃ of overnight incubation.Random choose list bacterium colony from flat board, carries out pcr amplification with the primer of each functional gene, selects positive colony.
10, the abduction delivering of long-chain-acyl group-coenzyme A synthetic enzyme recombinant bacterium E. coli BL21/pET-28a/unsC
By being accredited as positive mono-clonal, be inoculated in the LB liquid nutrient medium that 5mL contains Kan resistance 37 ℃, 250r/min overnight incubation.Get 1mL culture, in the LB liquid nutrient medium that contains Kan resistance in 50mL of being transferred, 37 ℃, 250r/min are cultured to cell concentration OD600 and are about 0.6～0.8 left and right.In culture, add respectively certain density IPTG inducing culture 8h.Collecting thalline surveys for electrophoretic analysis and enzyme biopsy.
11, long-chain-acyl group-coenzyme A synthetic enzyme recombinant bacterium E. coli BL21/pET-28a/unsC expression product SDS-PAGE analyzes
With the recombinant bacterium that proceeds to the E. coli BL21 bacterium of empty carrier and do not add inductor IPTG in contrast.Be accredited as positive recombinant bacterium after IPTG inducing culture certain hour, get 0.5mL inducing culture thing, centrifugal collection thalline, be resuspended in 50 μ L distilled water, add 50 μ L sample-loading buffers, after mixing, boil 10min, carry out SDS-PAGE electrophoretic analysis, " A " swimming lane in Figure 10 is recombinant bacterium E. coli BL21/pET-28a/unsC
1long-chain-acyl group-coenzyme A synthetic enzyme the unsC expressing
1the SDS-PAGE figure of (through its aminoacid sequence of sequence verification as shown in SEQ ID No.1), " B " swimming lane is recombinant bacterium E. coli BL21/pET-28a/unsC
2long-chain-acyl group-coenzyme A synthetic enzyme the unsC expressing
2the SDS-PAGE figure of (through its aminoacid sequence of sequence verification as shown in SEQ ID No.3).
12, the protein-active of long-chain-acyl group-coenzyme A synthetic enzyme recombinant bacterium E. coli BL21/pET-28a/unsC detects
(1) long-chain-acyl group-coenzyme A synthetic enzyme unsC
1protein-active detects:
Enzyme liquid preparation: the recombinant bacterium E. coli BL21/pET-28a/unsC that takes collection
1phosphate buffered saline buffer for 0.5g (50mM, pH8.0) 15mL suspends, ultrasonication (power 350W, broken 2s, interval 2s, common ultrasonication 5min).
Long-chain-acyl group-coenzyme A synthetic enzyme unsC transformation system: transform in bottle and add E. coli BL21/pET-28a/unsC at 50mL
1ultrasonication thalline 10mL, 0.1g palm fibre eleostearic acid, 0.1g ATP, 0.1gCoA, 30 ℃, 150r/min conversion, after conversion finishes, centrifuging and taking supernatant is standby with subsequent detection.
Detection method: GC conditions: 30m * 0.32mm * 0.25mm fused-silica capillary column; 190 ℃ of post initial temperature post initial temperature, insulation 1min, is warming up to 230 ℃ with 6 ℃/min, then constant temperature; 250 ℃ of vaporizer temperature; Carrier gas is high-purity He (99.999%); Before post, press 62.6KPa; Flow rate of carrier gas 1.4mL/min; Sample size 1 μ L; Splitting ratio 60:1.Mass spectrum condition: ion source is EI source; 230 ℃ of ion source temperatures; 150 ℃ of quadrupole temperature; Electron energy 70eV; 260 ℃ of interface temperature; Solvent delay 2min; Mass range 10-550u.
Through above-mentioned chromatographic condition, detect and calculate long-chain-acyl group-coenzyme A synthetic enzyme recombinant bacterium E. coli BL21/pET-28a/unsC
1expressed long-chain-acyl group-coenzyme A synthetic enzyme unsC
1high specific enzyme live (Specific Activity) be 10.8 mol/min/mg, substrate conversion efficiency 75.86%.
(2) long-chain-acyl group-coenzyme A synthetic enzyme unsC
2protein-active detects:
Enzyme liquid preparation: the recombinant bacterium E. coli BL21/pET-28a/unsC that takes collection
2phosphate buffered saline buffer for 0.5g (50mM, pH8.0) 15mL suspends, ultrasonication (power 350W, broken 2s, interval 2s, common ultrasonication 5min).
Long-chain-acyl group-coenzyme A synthetic enzyme unsC
2transformation system: transform in bottle and add E. coli BL21/pET-28a/unsC at 50mL
2ultrasonication thalline 10mL, 0.1g palm fibre eleostearic acid, 0.1g ATP, 0.1gCoA, 30 ℃, 150r/min conversion, after conversion finishes, centrifuging and taking supernatant is standby with subsequent detection.
Detection method: GC conditions: 30m * 0.32mm * 0.25mm fused-silica capillary column; 190 ℃ of post initial temperature post initial temperature, insulation 1min, is warming up to 230 ℃ with 6 ℃/min, then constant temperature; 250 ℃ of vaporizer temperature; Carrier gas is high-purity He (99.999%); Before post, press 62.6KPa; Flow rate of carrier gas 1.4mL/min; Sample size 1 μ L; Splitting ratio 60:1.Mass spectrum condition: ion source is EI source; 230 ℃ of ion source temperatures; 150 ℃ of quadrupole temperature; Electron energy 70eV; 260 ℃ of interface temperature; Solvent delay 2min; Mass range 10-550u.
Through above-mentioned chromatographic condition, detect and calculate, we draw to draw a conclusion: long-chain-acyl group-coenzyme A synthetic enzyme recombinant bacterium E. coli BL21/pET-28a/unsC
2expressed long-chain-acyl group-coenzyme A synthetic enzyme unsC
2high specific enzyme live (Specific Activity) be 10.8 mol/min/mg, substrate conversion efficiency 79.53%.
Claims (5)
1. long-chain-acyl group-coenzyme A the synthetic enzyme that participates in brown eleostearic acid anabolism hexadecyl-coenzyme A, its aminoacid sequence is as shown in SEQ ID No.1.
2. long-chain-acyl group as claimed in claim 1-coenzyme A synthetic enzyme is prepared the application in hexadecyl-coenzyme A at biocatalysis palm fibre eleostearic acid.
3. the gene of long-chain-acyl group-coenzyme A synthetic enzyme described in the claim 1 of encoding.
4. gene as claimed in claim 3, is characterized in that the nucleotide sequence of described gene is as shown in SEQ ID No.2.
5. the application of gene as claimed in claim 4 in building the genetic engineering bacterium of can biocatalysis palm fibre eleostearic acid preparing hexadecyl-coenzyme A.
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| CN1584033A (en) * | 2004-06-03 | 2005-02-23 | 上海交通大学 | Taxaceae 3-hydroxy-3-methylpentadiacyl cozymase A synthetic zymoprotein coding sequence |
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