CN102978177B - Cordyceps sinensis delta-5-desaturase used in anabolism of eicosapentaenoic acid, and gene and application thereof - Google Patents
Cordyceps sinensis delta-5-desaturase used in anabolism of eicosapentaenoic acid, and gene and application thereof Download PDFInfo
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- CN102978177B CN102978177B CN201210447392.7A CN201210447392A CN102978177B CN 102978177 B CN102978177 B CN 102978177B CN 201210447392 A CN201210447392 A CN 201210447392A CN 102978177 B CN102978177 B CN 102978177B
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- 239000012138 yeast extract Substances 0.000 description 1
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Abstract
The invention relates to a delta-5-desaturase from a Corbrin-production bacterium cordyceps sinensis hirsutella sinensis. The delta-5-desaturase participates in eicosapentaenoic coenzyme A anabolism started from eicosatetraenoic coenzyme A. The invention also relates to a gene coding the enzyme, and an application thereof. The amino acid sequence of the delta-5-desaturase has more than 90% homology with a sequence represented by SEQ ID No.1 or SEQ ID No.3. From the principle, the invention carries out detailed researches upon the metabolic pathway of eicosatetraenoic coenzyme A to synthesizing the eicosapentaenoic coenzyme A. A cloned DNA comprising the nucleotide sequence provided by the invention can be transferred to engineering bacteria through methods of transduction, transformation, and combined transferring. Through the regulation upon eicosapentaenoic coenzyme A biosynthetic gene expression, high expression of a host delta-5-desaturase is provided, an effective approach is provided for increasing eicosapentaenoic acid yield. The application has an important application prospect.
Description
(1) technical field
The present invention relates to a kind of participation Eicosatetraenoic coenzyme A from " hundred makes " production bacterium Cordyceps sinensis China pilose spore to set out the △-5-desaturase (△-5 desaturase) of anabolism eicosa-pentaenoic coenzyme A, the gene of this enzyme of encoding and application thereof.Eicosa-pentaenoic coenzyme A generates timnodonic acid through hydrolysis again.
(2) background technology
Cordyceps sinensis (Cordyceps sinensis (Berk.) Sacc.) is that Cordyceps fungus colonizes in the stroma on lepidopteran (Lepidoptera) Hepialidae insect (Hepialus armoricanus Oberthur) larva and the complex body (comprising stroma and polypide) on larva corpse.Cordyceps sinensis is traditional fungi herb resource that a class is treasured, and has the feature of meta-bolites and diverse biological activities, shows huge application and development prospect at biomedicine field.Cordyceps sinensis extensively, obviously receives much concern with its multiple medicinal efficacy, worldwide enjoys high praise.The traditional Chinese medical science is thought, Cordyceps sinensis enters lung kidney two warp, can tonifying lung cloudy, again can kidney-replenishing, 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, and phthisical cough phlegm blood, spontaneous sweating etc. are unique a kind of Chinese medicine that can balance simultaneously, regulate 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 in the actual production such as artificial culture, liquid fermenting, use the Cordyceps fungus in imperfect stage, thus the qualification of Anamorph of Cordyceps Sinensis is extremely important.Chinese scholars is done a lot of work in 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, and price is high.Wild cordyceps owing to restricting by factors such as growing environments, scarcity of resources.Owing to making little progress on artificial culture in recent years, the research of wild cordyceps surrogate focused mostly on liquid fermenting.Utilizing liquid submerged fermentation to cultivate Cordyceps mycelium, extract or fermented liquid, is a kind of effective way solving Cordyceps sinensis medicine source.Chinese caterpillar fungus fermentation produces Chinese caterpillar fungus substitute, both can these precious resources of available protecting Chinese caterpillar fungus, and the restriction that climate, geographical environment and Chinese caterpillar fungus parasitic conditions are not strict again, is suitable for industrialization scale operation.The substitute produced is as also similar to natural cs with drug effect in its composition of mycelium, is thus 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, prove with natural cs chemical constitution, pharmacological action basically identical, natural cs can be replaced 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 techniques, in worm grass product research and development, progressively turn to deeper functional metabolic Study on product by the direct utilization of Chinese caterpillar fungus raw material or crude extract.Large quantifier elimination is done to Chinese caterpillar fungus meta-bolites both at home and abroad, meta-bolites mainly comprises several large compounds such as nucleosides, polysaccharide, polypeptide, sterol, and wherein the representative research of functional metabolic product in biosynthesizing, pharmacological action etc. such as purines nucleosides, Cordyceps polysaccharide, N.F,USP MANNITOL wins initial success.
Unsaturated fatty acids refers to the lipid acid containing one or more double bond in molecule, and its fusing point comparatively saturated fatty acid is low.Unsaturated fatty acids is a kind of lipid acid forming body fat, and the lipid acid of needed by human, unsaturated fatty acids, according to the difference of double bond number, is divided into monounsaturated fatty acids and polyunsaturated fatty acid two kinds.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 the function etc. of human defensive system, in addition it can also be got rid of " rubbish " unnecessary in human body, namely owing to taking the photograph the superabundant fat that the excessive saturated fatty acid of people is formed, thus reaches the object of fat-reducing.Therefore, its potential medical pharmaceutical use receives the extensive concern in the world, causes the great attention of the industry such as food, medical even makeup.
Yung-Sheng in 1999 draws the △ 6 and △ 12 fatty acid dehydrogenase gene that have cloned Mortierella alpina (Mortiere Uaalpina) and expresses in yeast saccharomyces cerevisiae.2004, △ 3 desaturase in tung oil tree was proceeded to yeast by the people such as Dyer, successfully obtained and produced linolenic yeast.△ 12 desaturase of blue or green for algae bacterium (Cyanobacterium) is proceeded to potato by the people such as Maali-Amiri in 2007, successfully detects that potato fatty acid component there occurs considerable change.2008, △ 6 desaturase of volume branch Mucor turned in people's transgene tobacco by the people such as Hao, have successfully been obtained the bacterial strain of high yield gamma-linolenic acid.In addition, the relevant gene of many fatty acid desaturases is also had to be cloned and Transformation Application.Because most of desaturase is embrane-associated protein, its separation and purification is very difficult, and the desaturase that separation and purification is also identified cans be counted on one's fingers, and the research of the overwhelming majority is carried out around delta 8 desaturase genes and expression regulation thereof.
At present, the unsaturated fatty acids applied produces bacterium based on subtilis, 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 above deficiency that exists and the technical issues that need to address, the enzyme of bacterium Cordyceps sinensis China pilose spore anabolism timnodonic acid is produced to " hundred make " and encoding gene is furtherd investigate, provide " hundred makes " and produce bacterium Cordyceps sinensis China pilose spore participation Eicosatetraenoic coenzyme A and to set out the enzyme of anabolism eicosa-pentaenoic coenzyme A, encoding gene and application thereof.
The technical solution used in the present invention is:
Participate in Eicosatetraenoic coenzyme A to set out the △-5-desaturase of anabolism eicosa-pentaenoic coenzyme A, with sequence shown in SEQ ID No.1 or SEQ ID No.3, there is more than 90% homology; This enzyme can prepare corresponding eicosa-pentaenoic coenzyme A by catalysis Eicosatetraenoic coenzyme A, and eicosa-pentaenoic coenzyme A generates timnodonic acid through the effect of Perhydrolase again.Due to the singularity of aminoacid sequence; the fragment of any peptide protein containing aminoacid sequence shown in SEQ ID NO.1 or SEQ ID No.3 or its variant; 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 sequences homology, more than 90%, all belong to the row of scope.Concrete described change can comprise amino acid whose disappearance, insertion or replacement in aminoacid sequence; Wherein, the conservative property for variant changes, and the amino acid replaced 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 △-5-desaturase aminoacid sequence such as SEQ ID No.1(is designated as unsF
1albumen) or SEQ ID No.3 shown in (be designated as unsF
2albumen).
The present invention states △-5-desaturase and produces bacterium Cordyceps sinensis China pilose spore from " hundred make ".
As follows by the path that Eicosatetraenoic coenzyme A anabolism obtains corresponding eicosa-pentaenoic coenzyme A, eicosa-pentaenoic CoA hydrolase generates timnodonic acid:
The invention still further relates to described △-5-desaturase and prepare application in eicosa-pentaenoic coenzyme A at biocatalysis Eicosatetraenoic coenzyme A.
The invention still further relates to the encoding gene of above-mentioned △-5-desaturase, namely △-5-dehydrogenase gene, concrete, and this encoding gene can be the gene order with polynucleotide shown in SEQ ID NO:2 or SEQ ID No.4 with more than 90% homology.Due to the singularity of nucleotide sequence, shown in any SEQ ID NO:2 or SEQ ID No.4, the variant of polynucleotide, as long as itself and this polynucleotide have more than 90% homology, all belongs to the row of scope.The variant of described polynucleotide refers to a kind of polynucleotide sequence having one or more Nucleotide and change.The variant of these polynucleotide can make raw displacement varient or the varient of non-life, comprises and replaces varient, Deletion variants and insertion varient.As known in the art, allelic variant is the replacement form of polynucleotide, and it may be the replacement of polynucleotide, disappearance or insertion, but can not from the function of peptide protein changing in fact its coding.
Preferably, described △-5-dehydrogenase gene nucleotide sequence such as SEQ ID No.2(is designated as unsF
1gene) or SEQ ID No.4(be designated as unsF
2gene) shown in.
Described gene can be used for building can prepare the genetic engineering bacterium of eicosa-pentaenoic coenzyme A, to expand the output of timnodonic acid or derivatives thereof by biocatalysis Eicosatetraenoic coenzyme A.
Main points of the present invention there are provided the nucleotide sequence shown in the aminoacid sequence shown in SEQ ID NO:1 or 3 and SEQ ID NO:2 or 4, when this aminoacid sequence known and nucleotide sequence, the acquisition of this aminoacid sequence and nucleotide sequence, and related vector, host cell acquisition, 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 Eicosatetraenoic coenzyme A synthesis eicosa-pentaenoic coenzyme A pathways metabolism principle, the cloned DNA comprising nucleotide sequence provided by the present invention can be used for proceeding in engineering bacteria by the method for transduction, conversion, Conjugative tiansfer, by regulating the expression of eicosa-pentaenoic coenzyme A biosynthesis gene, give the host △-5-high expression level of desaturase, for the output expanding timnodonic acid or derivatives thereof provides effective way, there is major application prospect.
(4) accompanying drawing explanation
Fig. 1 is the denaturing formaldehyde gel electrophorogram that " hundred make " produces bacterium Cordyceps sinensis China pilose spore total serum IgE;
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 △-5-dehydrogenase 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 F physical map;
Fig. 8 is recombinant expression plasmid pET-28a/uns F building process schematic diagram;
Fig. 9 is recombinant expression plasmid pET-28a/uns F physical map;
Figure 10 is the SDS-PAGE figure of △-5-apodehydrogenase.
(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 carry out separation screening, obtain L0106 bacterial strain, and be China pilose spore (Hirsutella Sinensis) through this bacterial strain of strain identification, this culture presevation is in China typical culture collection center, deposit number is CCTCC No:M 2011278, discloses in the patent CN102373190A of previously application.
By this strain inoculation in inclined-plane, (this is the liquid formulations before solidification to culture medium prescription, by following proportions well after bevel again) be glucose 2.0%(w/v, 1% represents in 100mL substratum containing 1g, down together), 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%, surplus is water, cultivates 25 days at 12 ~ 16 DEG C; Then by strain inoculation in 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, temperature 12 ~ 16 DEG C is cultivated 25 days, after cultivation terminates aseptically, carries out solid-liquid separation, and solid is placed in sterilized equipment, for subsequent use.
Embodiment 2: " hundred make " produces the extraction of bacterium Cordyceps sinensis China pilose spore total serum IgE
Total serum IgE is extracted with TRIzol reagent, step is specially: 1) liquid nitrogen grinding: get the new fresh thalli of 1g and put into mortar, repeatedly adding liquid nitrogen is fully ground to Powdered, be dispensed in the 1.5mL centrifuge tube of precooling, add 1mL TRIzol reagent, mixing, leaves standstill 5min on ice, nucleic acid-protein mixture is separated completely.2) RNA is separated: add 0.2mL chloroform, firmly concussion mixing 15s, and leave standstill 2 ~ 3min on ice, 4 DEG C, the centrifugal 15min of 12000rpm, layering, gets upper strata aqueous phase, about 600 μ L.3) RNA precipitation: add 500 μ L Virahols, leaving standstill 10min on ice, 4 DEG C, the centrifugal 10min of 12000rpm, abandon supernatant.4) RNA washing: add 1mL 75%(v/v) ethanol, hangs precipitation, leaves standstill 10min on ice, 4 DEG C, the centrifugal 15min of 7500rpm; Repeat washing step above, then wash one time.5) RNA is dissolved: be placed in by centrifuge tube and open 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
After extracting sample total serum IgE, with Oligo(dT) enrichment with magnetic bead mRNA.Add fragmentation buffer and mRNA is broken into short-movie section (200 ~ 700bp), take mRNA as template, Article 1 cDNA chain is synthesized with hexabasic base random primer (random hexamers), then Article 2 cDNA chain is synthesized, do end reparation after adding EB buffer solution elution through QiaQuick PCR kit purifying, add polyA and connect sequence measuring joints again, then clip size selection is carried out with agarose gel electrophoresis, finally carry out pcr amplification, the sequencing library Illumina GA IIx built up checks order.The raw image data obtained that checks order is converted into sequence data through base calling, i.e. raw data or raw reads.Only containing the reads of adaptor sequence in removing primitive sequencer reads, standby with subsequent analysis.
Embodiment 4: " hundred make " production bacterium Cordyceps sinensis China pilose spore RNA is short reads sequence assembling
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.) do transcript profile and from the beginning assemble.First the reads with certain length overlap is linked to be the longer Contig fragment not containing N by SOAPdenovo.Then Contig is returned in reads comparison, determine from the distance between the different Contig of same transcript and these Contig by paired-end reads, these Contig connect together by SOAPdenovo, and middle unknown nucleotide sequence N represents, so just obtains Scaffold.Utilize paired-end reads to do filling-up hole process to Scaffold further, 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 blastx comparison (evalue<0.00001), get the sequence direction that the best albumen of comparison result determines Unigene.If the comparison result between different sink is contradictory, then press nr, Swiss-Prot, the priority of KEGG and COG determines 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 its coding region and determine the direction of sequence.For determining that the Unigene in sequence direction provides the sequence in its direction from 5' to 3', for determining the sequence that the Unigene in sequence direction provides composite software and obtains.
Embodiment 5: " hundred make " produces bacterium Cordyceps sinensis China pilose spore Unigene functional annotation
Functional annotation information provides the protein function annotation of Unigene, Pathway annotation, COG functional annotation and Gene Ontology(GO) functional annotation.First, by blastx by Unigene sequence alignment to albumen database nr, Swiss-Prot, KEGG and COG(evalue<0.00001), obtain the albumen with given Unigene with highest serial similarity, thus obtain the protein function annotation information of this Unigene.The Pathway annotation of Unigene can be obtained further according to KEGG annotation information.Compared by Unigene and COG database, the function that prediction Unigene is possible also does 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): 3674-3676.) the GO annotation information of Unigene is obtained.After obtaining 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.) GO functional classification statistics is done, from the gene function distribution characteristics being macroscopically familiar with these species to all Unigene.
Embodiment 6: " hundred make " is produced bacterium Cordyceps sinensis China pilose spore timnodonic acid 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 annotated is that " hundred make " detected produces bacterium Cordyceps sinensis China pilose spore timnodonic acid pathways metabolism relevant enzymes, as can be seen from the figure, the △-5-dehydrogenase 2 Unigene from the corresponding eicosa-pentaenoic coenzyme A of each Eicosatetraenoic coenzyme A synthesis is detected.By the ORF Finder software on-line checkingi in NCBI, have found the open reading frame (SEQ ID No.2, No.4) of this gene and obtain corresponding protein sequence (SEQ ID No.1, No.3).
Embodiment 7: " hundred make " produces bacterium Cordyceps sinensis China pilose spore △-5-dehydrogenase gene design of primers
Use GENE RUNNER primer-design software according to predicting each gene open proofreading dna primers obtained, △-5-the dehydrogenase gene of bacterium China pilose spore anabolism timnodonic acid is produced for clone's " hundred make ", primer by Shanghai Sheng Gong biotechnology company limited synthesize, primer sequence as follows listed by:
UnsF
1gene: forward primer 5 ' ACAGAATTCATGCCTGAATGCGCCCGTTC 3 '
Reverse primer 5 ' ATAAAGCTTTCACGTCGCAAGCTGGCGATG 3 '
UnsF
1mrna length is 363bp.
UnsF
2gene: forward primer 5 ' ACGGAATTCATGTGGCTTGAGATTAAGCAG 3 '
Reverse primer 5 ' AGCAAGCTTTCAGTAGATGAGAGGGTGATGG 3 '
UnsF
2mrna length is 225bp.
Embodiment 8: " hundred make " produces the preparation of bacterium Cordyceps sinensis China pilose spore cDNA first chain
After the method first provided according to embodiment 1 turns out sutella sinensis fermented mycelium, the method provided according to embodiment 2 again carries out the extraction of total serum IgE to China pilose spore, carry out by following the synthesis that " hundred make " produces bacterium Cordyceps sinensis China pilose spore cDNA first chain, for follow-up each gene clone experiment after obtaining total serum IgE.
Adopt PrimeScript 1st Strand cDNA Synthesis Kit test kit (TaKaRa) reverse transcription synthesis cDNA first chain from Total RNA, experimental procedure is as follows:
1) in Microtube pipe, following mixed solution is prepared.
2) sex change, annealing operation are conducive to the sex change of template ribonucleic acid and the specificity annealing of reverse transcription primer and template, and can improve reverse transcription reaction efficiency, so carry out sex change, annealing reaction in PCR instrument, condition setting is as follows:
65℃，5 min
3) annealing terminates the rear centrifugal several seconds mixed solution of template ribonucleic acid/primer etc. is gathered in bottom Microtube pipe.
4) in above-mentioned Microtube pipe, following inverse transcription reaction liquid is prepared.
5) in PCR instrument, reverse transcription reaction is carried out by following condition.
42℃ 15～30 min
70℃ 15 min
Generalized case, a PolyA structure is had at eukaryote mRNA 3 ' end, the quantity of A base is not at ten to hundreds of etc., utilize this structure can utilize Oligo (dT) primer, under the effect of ThermoScript II, take mRNA as templated synthesis cDNA first chain, the present invention adopts the sequence in the dT region developed alone by TaKaRa (providing in PrimeScript 1st Strand cDNA Synthesis Kit) to be primer, if the mRNA integrity obtained is better, cDNA first chain of all zymoprotein encoding genes in species so can be obtained by process of reverse-transcription.
Embodiment 9: " hundred make " produces the detection of the clone of bacterium Cordyceps sinensis China pilose spore anabolism timnodonic acid functional gene △-5-desaturase unsF gene, expression and protein vigor
1, the pcr amplification of △-5-desaturase unsF gene
With cDNA first chain obtained in embodiment 8 for template, with the unsF of synthesis in embodiment 7
1gene primer: 5 ' ACA GAA TTC ATG CCT GAA TGC GCC CGT TC3 ' and 5 ' ATA AAG CTT TCA CGT CGC AAG CTG GCG ATG3 ', unsF
2gene primer: 5 ' ACG GAA TTC ATG TGG CTT GAG ATT AAG CAG3 ' and 5 ' AGC AAG CTT TCA GTA GAT GAG AGG GTG ATG G3 ' carries 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, △-5-desaturase unsF gene PCR product detected through gel electrophoresis
Concrete detection method is: 1) make it be uniformly dissolved the sepharose microwave-oven-heating of prepare 0.9%; 2) get 15mL gel, when gel is cooled to about 50 DEG C, add 1 μ L staining fluid Gold view, pour into after mixing on treatments of Electrophoretic Slab Gels, after removing bubble, insert point sample comb; 3), after gel sets, the careful point sample that takes out is combed, and offset plate is put into electrophoresis chamber (loading wells one end is near the negative pole of electrophoresis chamber), adds TAE electrophoretic buffer in electrophoresis chamber; 4) get 5 μ L samples and then add 6 × Loading Buffer 1.5 μ L and ddH
2o 4 μ L uses liquid-transfering gun loading after mixing, and 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, start electrophoresis, maximum voltage is no more than 5 V/cm; 7) electrophoresis can be stopped when sample ran 2/3 of offset plate; 8), after cutting off the electricity supply, gel taken out and puts into the observation of gel imaging instrument, take pictures.
Transcript profile order-checking prediction △-5-desaturase unsF
1the size of gene is 363bp, unsF
2the size of gene is 225bp, and agarose gel electrophoresis result shows that Successful amplification has gone out △-5-desaturase unsF
1gene, size is about 360bp, unsF
2gene, size is about 220bp.Fig. 5 is that " hundred make " produces bacterium China pilose spore anabolism eicosa-pentaenoic coenzyme A functional gene PCR primer gel electrophoresis figure.
3, △-5-desaturase unsF gene PCR product add base A process and purifying
Because Pfu archaeal dna polymerase PCR primer end is flush end, connect so just can be used for carrier T also need to carry out adding base A process, purifying after glue reclaims after.It is as follows that glue recovery product adds base A system:
In PCR instrument, 72 DEG C add A base 20 min, finally purify with AxyPrep PCR cleaning agents box.
4, the connection of △-5-desaturase unsF gene and cloning vector
Cloning vector pMD18-T Vector is purchased from TaKaRa company (TaKaRa code D101A), and its physical map is shown in Fig. 6, by △-5-desaturase unsF
1, unsF
2gene is connected construction recombination plasmid pMD18-T/unsF with cloning vector
1, pMD18-T/unsF
2, physical map is shown in Fig. 7, linked system and condition of contact as follows.
Linked system:
Condition of contact: 16 DEG C, 16h; Deactivation: 65 DEG C, 15min.
5, the conversion of △-5-desaturase recombinant plasmid pMD18-T/unsF
Recombinant plasmid pMD18-T/unsF is proceeded to the recombinant bacterium E. coli JM109/pMD18-T/unsF building in intestinal bacteria E. coli JM109 and carry △-5-desaturase unsF gene, concrete steps are: 1) go in competent cell E. coli JM109 by 10 μ L reaction systems, ice bath 30min; 2) thermal shock: 42 DEG C, 90s; 3) ice bath: 2-3min; 4) 800 μ L liquid LB are added, 37 DEG C, 250rpm, 1h; 5) spread plate (containing Amp resistance); 6) 37 DEG C of incubator overnight incubation.
6, the screening of the positive recombinant bacterium of △-5-desaturase E. coli JM109/pMD18-T/unsF
Bacterium colony PCR can extract genomic dna, and directly with the DNA exposed after thalline pyrolysis for template carries out pcr amplification, the method is easy and simple to handle, quick, can Rapid identification bacterium colony be whether positive bacterium colony containing object plasmid, comparatively common in conversion qualification.In experiment, carrying out bacterium colony PCR by being inoculated into single bacterium colony corresponding in liquid nutrient medium, whether proceeding to goal gene to verify.First, add in the 1.5mL centrifuge tube containing 50 μ L sterilized waters with toothpick picking list bacterium colony, boiling water bath 30min, then centrifugal using supernatant as template, carry out pcr amplification, PCR program setting is Taq enzyme amplification general procedure.The agarose gel electrophoresis of 0.9% is finally adopted to detect bacterium colony PCR primer.
7, △-5-desaturase recombinant plasmid pMD18-T/unsF
1and pMD18-T/unsF
2order-checking
After the positive recombinant bacterium LB liquid medium overnight incubation that bacterium colony PCR is detected, 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 is to recombinate to pMD18-T/unsF respectively
1and pMD18-T/unsF
2in
8, △-5-desaturase recombinant expression plasmid pET-28a/unsF
1and pET-28a/unsF
2structure
Experimental basis foreign gene is in the principle of expression in escherichia coli, and expression vector pET-28a and △-5-desaturase unsF gene restriction enzyme site comparison situation, determine EcoR I and Hind III double enzyme site, and to recombination bacillus coli E. coli JM109/pMD18-T/unsF
1with E. coli JM109/pMD18-T/unsF
2carry out the cultivation of liquid LB test tube shaker, recombinant plasmid extraction.
The recombinant plasmid pMD18-T/unsF of △-5-desaturase unsF gene
1and pMD18-T/unsF
2and expression vector pET-28a use respectively EcoR I/Hind III restriction enzyme 37 DEG C respectively enzyme cut process 6h, it is as follows that enzyme cuts system:
EcoR I/Hind III double digestion system:
Enzyme is cut and is terminated rear 65 DEG C of deactivation 15min, then respectively with Axygen DNA gel recovery test kit carry out reclaiming, purifying.
△-5-desaturase unsF gene and expression vector pET-28a connect with T4 ligase enzyme 16 DEG C again and spend the night after double digestion, purifying, build recombinant expression plasmid pET-28a/unsF
1and pMD18-T/unsF
2, its building process is shown in Fig. 8, builds the recombinant expression plasmid pET-28a/unsF obtained
1, pET-28a/unsF
2fig. 9 is shown in by collection of illustrative plates.Linked system is composed as follows:
Linked system:
9, the conversion of △-5-desaturase recombinant expression plasmid and the screening of positive monoclonal
By heat-shock transformed in E. coli BL21 Host Strains for the expression plasmid built, be then applied on the LB agar plate containing kantlex (Kan) resistance, 37 DEG C of overnight incubation.From flat board, random choose list bacterium colony, carries out pcr amplification with the primer of each functional gene, selects positive colony.
10, the abduction delivering of △-5-desaturase recombinant bacterium
The mono-clonal being accredited as the positive is inoculated in 5mL to be contained in the LB liquid nutrient medium of Kan resistance, 37 DEG C, 250r/min overnight incubation.Get 1mL culture, transferred in 50mL to contain in the LB liquid nutrient medium of Kan resistance 37 DEG C, 250r/min is cultured to cell concentration OD600 and is about about 0.6 ~ 0.8.Certain density IPTG inducing culture 8h is added respectively in culture.Collect thalline for electrophoretic analysis and Enzyme activity assay.
11, △-5-desaturase recombinant bacterium expression product SDS-PAGE analyzes
With the E. coli BL21 bacterium proceeding to empty carrier and do not add inductor IPTG recombinant bacterium in contrast.Be accredited as positive recombinant bacterium after IPTG inducing culture certain hour, get 0.5mL Induced cultures, collected by centrifugation thalline, be resuspended in 50 μ L distilled water, add 50 μ L sample-loading buffers, boil 10min after mixing, carry out SDS-PAGE electrophoretic analysis, " A " swimming lane in Figure 10 is recombinant bacterium E. coli BL21/pET-28a/unsF
1△-5-desaturase the unsF expressed
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/unsF
2△-5-desaturase the unsF expressed
2the SDS-PAGE figure of (through its aminoacid sequence of sequence verification as shown in SEQ ID No.3).
12, the protein-active of △-5-desaturase recombinant bacterium detects
(1) Δ-5-desaturase unsF
1protein-active detect:
Prepared by enzyme liquid: the recombinant bacterium E. coli BL21/pET-28a/unsF taking collection
10.5g phosphate buffered saline buffer (50mM, pH8.0) 15mL suspends, ultrasonication (power 350W, broken 2s, interval 2s, altogether ultrasonication 5min).
△-5-desaturase unsF
1transformation system: transform in bottle at 50mL and add E. coli BL21/pET-28a/unsF
1ultrasonication thalline 10mL, 0.1g Eicosatetraenoic coenzyme A, 30 DEG C, 150r/min conversion, transform after terminating, and centrifuging and taking supernatant is standby with subsequent detection.
Detection method: GC conditions: 30m × 0.32mm × 0.25mm fused-silica capillary column; Post initial temperature post initial temperature 190 DEG C, insulation 1min, is warming up to 230 DEG C with 6 DEG C/min, then constant temperature; Vaporizer temperature 250 DEG C; Carrier gas is high-purity He (99.999%); Pressure 62.6KPa before post; Flow rate of carrier gas 1.4mL/min; Sample size 1 μ L; Splitting ratio 60:1.Mass Spectrometry Conditions: ion source is EI source; Ion source temperature 230 DEG C; Quadrupole temperature 150 DEG C; Electron energy 70eV; Interface temperature 260 DEG C; Solvent delay 2min; Mass range 10-550u.
Detect through above-mentioned chromatographic condition and calculate, draw to draw a conclusion: △-5-desaturase recombinant bacterium E. coli BL21/pET-28a/unsF
1expressed △-5-desaturase unsF
1high specific enzyme live (Specific Activity) be 8.2 mol/min/mg, substrate conversion efficiency is 83.95%.
(2) Δ-5-desaturase unsF
2protein-active detect:
Prepared by enzyme liquid: the recombinant bacterium E. coli BL21/pET-28a/ unsF taking collection
20.5g phosphate buffered saline buffer (50mM, pH8.0) 15mL suspends, ultrasonication (power 350W, broken 2s, interval 2s, altogether ultrasonication 5min).
△-5-desaturase unsF
2transformation system: transform in bottle at 50mL and add E. coli BL21/pET-28a/ unsF
2ultrasonication thalline 10mL, 0.1g Eicosatetraenoic coenzyme A, 30 DEG C, 150r/min conversion, transform after terminating, and centrifuging and taking supernatant is standby with subsequent detection.
Detection method: GC conditions: 30m × 0.32mm × 0.25mm fused-silica capillary column; Post initial temperature post initial temperature 190 DEG C, insulation 1min, is warming up to 230 DEG C with 6 DEG C/min, then constant temperature; Vaporizer temperature 250 DEG C; Carrier gas is high-purity He (99.999%); Pressure 62.6KPa before post; Flow rate of carrier gas 1.4mL/min; Sample size 1 μ L; Splitting ratio 60:1.Mass Spectrometry Conditions: ion source is EI source; Ion source temperature 230 DEG C; Quadrupole temperature 150 DEG C; Electron energy 70eV; Interface temperature 260 DEG C; Solvent delay 2min; Mass range 10-550u.
Detect through above-mentioned chromatographic condition and calculate, draw to draw a conclusion: △-5-desaturase recombinant bacterium E. coli BL21/pET-28a/unsF
2expressed △-5-desaturase unsF
2high specific enzyme live (Specific Activity) be 7.8 mol/min/mg, substrate conversion efficiency is 82.83%.
Claims (5)
1. participate in a △-5-desaturase for Eicosatetraenoic coenzyme A synthesis eicosa-pentaenoic coenzyme A, its aminoacid sequence is as shown in SEQ ID No.1.
2. △-5-desaturase as claimed in claim 1 prepares the application in eicosa-pentaenoic coenzyme A at biocatalysis Eicosatetraenoic coenzyme A.
3. the gene of △-5-desaturase described in coding claim 1.
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. gene as claimed in claim 3 is building and can prepare application in the genetic engineering bacterium of eicosa-pentaenoic coenzyme A by biocatalysis Eicosatetraenoic coenzyme A.
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