CN104130999A - Uridylic acid synthetase from cordyceps sinensis hirsutella sinensis, coding gene and applications thereof - Google Patents

Abstract

The invention relates to an enzyme, which can produce uridylic acid from orotidylic acid through anabolism and is derived from a "Bailing" producing strain of cordyceps sinensis hirsutella sinensis, a gene coding the enzyme, and applications thereof. The enzyme is uridylic acid synthetase, the amino acid sequence of the enzyme has 90% or more of homology of the amino acid sequence represented by the SEQ ID No.1, SEQ ID No.3, or SEQ ID No.5, and the coding gene of the enzyme has 90% or more of homology of the nucleotide sequence represented by SEQ ID No.2, SEQ ID No.4, or SEQ ID No.6. The metabolic pathway of synthesis of uridylic acid from orotidylic acid is carefully researched in theory. The cloned DNA of the provided nucleotide sequence can be transferred into engineering bacteria through transduction, conversion, and conjugal transfer. Through adjusting the expression of uridylic acid biosynthesis gene, high expression is endowed to the host uridylic acid, thus an effective way is provided for increasing the output of uridylic acid, and the synthetase has a significant application prospect.

Description

From uridylic acid synthetic enzyme, encoding gene and the application thereof of Cordyceps sinensis China pilose spore

The application is application number 2012105352864, and name is called the divisional application of Cordyceps sinensis China pilose spore uridylic acid synthetic enzyme, encoding gene and application patent application thereof

(1) technical field

The present invention relates to the uridylic acid synthetic enzyme (uridine monophosphate synthetase) that a participation orotidylic acid from " hundred make " production bacterium Cordyceps sinensis China pilose spore becomes metabolism uridylic acid, 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 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 various feature of meta-bolites and biological activity, shows huge application and development prospect at biomedicine field.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 the moon, 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 weakness, 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 production such as artificial culture, liquid fermenting is the Cordyceps fungus in imperfect stage, thereby the qualification 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, prove with natural cs chemical constitution, pharmacological action basically identical, 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, in to worm grass product research and development, progressively turn to deeper functional meta-bolites to study by the direct utilization of Chinese caterpillar fungus raw material or crude extract.Chinese caterpillar fungus meta-bolites is 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 representative functional meta-bolites such as purines nucleosides, Cordyceps polysaccharide, N.F,USP MANNITOL wins initial success in the research of the aspect such as biosynthesizing, pharmacological action.

Ucleosides is one of topmost active substance of Chinese caterpillar fungus, and wherein miazines nucleosides comprises cytidine(C and uridine.Uridine (uracil riboside) claim again uridine (uIidine), and another name is by snow riboside, dihydro-pyrimidin nucleosides, the 1-β-D-ribosyl uridylic of muttering.Cytidine(C (cytosine riboside) has another name called cytidine (cytidine), cytidine, 1-β-D-furans nucleosides cytosine(Cyt).Pyrimidine nucleoside is multiduty nucleosides, can be used as the additive of healthcare products and food.It also becomes as beauty treatment and anti-ultraviolet radiation makeup the requisite that people live gradually, as medicine, clinical application is in nervus centralis, uropoiesis, metabolism and many-sided disease such as cardiovascular, in the U.S., nucleic acid medicine has shown irreplaceable effect at antiviral, anti-tumor aspect in recent years.

Research shows that cytidine is mainly for the production of intermediate antitumor, antiviral, along with going deep into of antiviral, antitumor, treatment AIDS-treating medicine development, the demand of natural pyrimidine nucleoside is strengthened.Cytidine is as pyrimidine nucleoside, mainly for the production of intermediate antitumor, antiviral, it is the main raw material of manufacturing the medicines such as cytosine arabinoside (Ara-CR), cyclotidine (CycloC), cytidine (CTP), cytidine diphosphate (CDP-Choline).

Uridine is a kind of natural nucleosides, is the precursor of every other pyrimidine nucleotide, and uridylic acid is one of 4 kinds of main mononucleotides that form nucleus ribosomal ribonucleic acid, has pyrimidine ring structure, and it is synthetic that it participates in glycogen, contributes to improve the hypoxia-bearing capability of cell.It is also the composition that forms zooblast nucleic acid, can improve body antibody horizontal, and uridine and derivative thereof can also be used to block various cancer cells and viral gene synthesizes, treatment cancer and the disease being caused by virus.

Due to the important physiological action of pyrimidine nucleoside and analogue thereof, related microorganism is produced the existing a lot of research of miazines nucleosides both at home and abroad.But, as the Cordyceps fungus of important anabolism miazines nucleosides, 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 miazines nucleosides 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 enzyme and the encoding gene thereof of bacterium Cordyceps sinensis China pilose spore anabolism uridylic acid and further investigate, the enzyme (being uridylic acid synthetic enzyme), encoding gene and the application thereof that provide " hundred make " production bacterium Cordyceps sinensis China pilose spore participation orotidylic acid to set out anabolism uridylic acid.

The technical solution used in the present invention is:

The invention provides and a kind of produce the set out Cordyceps sinensis China pilose spore uridylic acid synthetic enzyme of anabolism uridylic acid of bacterium Cordyceps sinensis China pilose spore participation orotidylic acid from " hundred makes ": described enzyme has the above homology of aminoacid sequence 90% shown in aminoacid sequence shown in aminoacid sequence shown in SEQ ID No.1, SEQ ID No.3 or SEQ ID No.5, preferred described enzyme amino acid sequence is that pynC1 albumen, the sequence of SEQ ID No.1 is that pynC2 albumen or the sequence of SEQ ID No.3 is the pynC3 albumen of SEQ ID No.5; This enzyme can be prepared uridylic acid by catalysis orotidylic acid.Due to the singularity of aminoacid sequence; any fragment or its variant that contains the peptide protein of aminoacid sequence shown in SEQ ID No.1, SEQ ID No.3 or SEQ ID No.5; 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, change for the conservative property 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.

The invention still further relates to described uridylic acid synthetic enzyme and prepare the application in uridylic acid at biocatalysis orotidylic acid.

Further, described is applied as: the broken mixed solution taking the wet thallus containing the acquisition of Cordyceps sinensis China pilose spore uridylic acid synthetic enzyme thalline fermentation culture after cytoclasis is as catalyzer, taking orotidylic acid as substrate, in the transformation system that the buffered soln that is 6.5～8.5 in pH forms, conversion reaction 2～3h under 30 DEG C, 150rpm condition, after reaction finishes, by reacting liquid filtering, get filtrate and be the crude product containing uridylic acid, described crude product separation and purification obtains uridylic acid.

Further, the starting point concentration of described substrate is 10g/L, and the volumetric usage of described catalyzer is 100mL/g substrate, and the dry mycelium concentration of described catalyzer is 6.7mg/mL.

The path that is obtained uridylic acid by orotidylic acid anabolism is shown below:

The preparation method of described catalyzer is: Cordyceps sinensis China pilose spore uridylic acid synthetic enzyme recombinant bacterium is inoculated in the LB liquid nutrient medium that 5mL contains Kan resistance (50mg/L) to 37 DEG C, 250r/min overnight incubation.Get 1mL culture, transferred in the fresh LB liquid nutrient medium that contains Kan resistance of 50mL 37 DEG C, 250r/min are cultured to cell concentration OD600 and are about 0.6～0.8 left and right, to the IPTG inducing culture 8h that adds finite concentration (240mg/ml) in culture, getting inducing culture liquid filters, collect wet thallus, take wet thallus phosphate buffered saline buffer (50mM, pH8.0) 15mL suspension for 0.5g of collection, the thalline mixed solution obtaining after ultrasonication (power 350W, broken 2s, interval 2s, common ultrasonication 5min) is as catalysis enzyme.

The invention still further relates to the encoding gene of above-mentioned Cordyceps sinensis China pilose spore uridylic acid synthetic enzyme, described gene has more than 90% homology of nucleotide sequence shown in nucleotide sequence shown in nucleotide sequence shown in SEQ ID No.2, SEQ ID No.4 or SEQ ID No.6, and preferred described gene order is that pynC1 gene, the sequence of SEQ ID No.2 is that pynC2 gene or the sequence of SEQ ID No.4 is the pynC3 gene of SEQ ID No.6.Due to the singularity of nucleotide sequence, the variant of polynucleotide shown in any SEQ ID NO:2, SEQ ID No.4 or SEQ ID No.6, 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 peptide protein of its coding.

The invention still further relates to described Cordyceps sinensis China pilose spore uridylic acid synthetase-coding gene in the application building in the genetic engineering bacterium of can biocatalysis orotidylic acid preparing uridylic acid, to expand the output of uridylic acid or derivatives thereof.

Being applied as preferably: build the recombinant vectors that contains described Cordyceps sinensis China pilose spore uridylic acid synthase gene, described recombinant vectors is converted in intestinal bacteria, the recombination engineering bacteria obtaining carries out inducing culture, and nutrient solution separation and purification obtains the somatic cells that contains Cordyceps sinensis China pilose spore uridylic acid synthetic enzyme.

The enzyme work of Cordyceps sinensis China pilose spore uridylic acid synthetic enzyme of the present invention is defined as: under optimum condition (30 DEG C), can transform the enzyme amount of 1 micromole's orotidylic acid in 1 minute.

The bacterial strain that Cordyceps sinensis China pilose spore uridylic acid synthetic enzyme of the present invention and encoding gene thereof can be provided is China pilose spore (Hirsutella Sinensis) L0106, this culture presevation is at Chinese Typical Representative culture collection center, deposit number is CCTCC No:M2011278, in the patent CN102373190A of previously application, discloses.

Main points of the present invention have been to provide the nucleotide sequence shown in the aminoacid sequence shown in SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5 and SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6, 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 uridylic acid pathways metabolism of orotidylic acid principle, the cloned DNA that comprises nucleotide sequence provided by the present invention can be used for by transduction, transform, proceeds in engineering bacteria in conjunction with the method shifting, by regulating the expression of uridylic acid biosynthesis gene, give the high expression level of host's uridylic acid, for the output that expands uridylic acid or derivatives thereof provides effective way, there is major application prospect.

(4) brief description of the drawings

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 pyrimidine metabolic approach annotated map;

Fig. 3 is uridylic acid synthase gene pcr amplification product gel electrophoresis figure;

Fig. 4 is cloning vector pMD18-T Vector and expression vector pET-28a physical map;

Fig. 5 is restructuring cloned plasmids pMD18-T/pynC physical map;

Fig. 6 is recombinant expression plasmid pET-28a/pynC building process schematic diagram;

Fig. 7 is recombinant expression plasmid pET-28a/pynC physical map;

Fig. 8 is uridylic acid synthetase albumen SDS-PAGE figure.

(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, obtain 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:M2011278, 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, prepare afterwards bevel again in following ratio) 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, cultivates 25 days at 12～16 DEG C; 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 DEG C of cultivations of temperature 25 days, under aseptic condition, carry out solid-liquid separation after cultivation finishes, and solid (being wet thallus) is placed in to aseptic utensil, for subsequent use.

Embodiment 2: " hundred make " produces the extraction of the total RNA of bacterium Cordyceps sinensis China pilose spore

Extract total RNA with TRIzol reagent, 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 1mLTRIzol reagent, mix, leave standstill 5min on ice, nucleic acid-protein mixture is separated completely.2) RNA separates: add 0.2mL chloroform, firmly concussion mixes 15s, leaves standstill 2～3min on ice, 4 DEG C, the centrifugal 15min of 12000rpm, and layering, gets upper strata water, approximately 600 μ L.3) RNA precipitation: add 500 μ L Virahols, leave standstill 10min on ice, 4 DEG C, the centrifugal 10min of 12000rpm, abandon supernatant.4) RNA washing: add 1mL75% (v/v) ethanol, will precipitate and hang, leave standstill 10min, 4 DEG C, the centrifugal 15min of 7500rpm on ice; 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, obtain " hundred make " and produce the total RNA of bacterium Cordyceps sinensis China pilose spore.

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 the enrichment with magnetic bead mRNA with Oligo (dT).Add fragmentation buffer that mRNA is broken into short-movie section (200～700bp), taking mRNA as template, with the synthetic Article 1 cDNA chain of hexabasic base random primer (random hexamers), then synthetic Article 2 cDNA chain, do end reparation, add polyA and connect sequence measuring joints through QiaQuickPCR test kit purifying and after adding EB buffer solution elution again, then carry out clip size selection with agarose gel electrophoresis, 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 the longer Contig fragment that does not contain N.Then reads is compared back to Contig, determine from the distance between different Contig and these Contig of same transcript by paired-end reads, 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 processing to Scaffold, 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 best albumen of comparison result and determine the sequence direction of 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 software ESTScan (Iseli for Unigene, Jongeneel et al.ESTScan:a program for detecting, evaluating, and reconstructing potential coding regions in EST sequences[J] .In Proceedings of9th 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.Provide its sequence from 5' to 3' direction for the Unigene that can determine sequence direction, provide for the Unigene that cannot determine sequence direction 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 Gene Ontology (GO) functional annotation of Unigene.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.Can further obtain the Pathway annotation of Unigene according to KEGG annotation information.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): 3674-3676.) obtain the GO annotation information of 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, be familiar with the gene function distribution characteristics of these species from macroscopic view.

Embodiment 6: " hundred make " produced bacterium Cordyceps sinensis China pilose spore uridylic acid pathways metabolism and analyzed

Fig. 2 is the pyrimidine metabolic (map00240) in KEGG pathways metabolism annotation, the enzyme having annotated is that " hundred make " that detected produces bacterium Cordyceps sinensis China pilose spore pyrimidine metabolic approach relevant enzymes, as can be seen from the figure, detected from 3 Unigene of uridylic acid synthetic enzyme of the synthetic uridylic acid of orotidylic acid.Detect online by the ORF Finder software in NCBI, found out the open reading frame (SEQ ID No.2, SEQ ID No.4 and SEQ ID No.6) of this gene and obtained corresponding protein sequence (SEQ ID No.1, SEQ ID No.3 and SEQ ID No.5).

Embodiment 7: " hundred make " produces bacterium Cordyceps sinensis China pilose spore uridylic acid synthase gene design of primers

The gene open reading frame DNA sequence dna design primer that uses GENE RUNNER primer-design software to obtain according to prediction, produce the uridylic acid synthase gene of bacterium China pilose spore anabolism uridylic acid for clone's " hundred make ", primer is synthetic by Shanghai Sheng Gong biotechnology company limited, and primer sequence is listed as follows:

PynC1 gene: forward primer 5 ' AGAGAATTCATGTTGGCTGACGCTAGGGTC3 '

Reverse primer 5 ' ATAAAGCTTTCACCCTCTGCCGCACCAACTTG3 '

PynC1 mrna length is 414bp

PynC2 gene: forward primer 5 ' ATTGGATCCATGCAGTACACTGGCGGCTC3 '

Reverse primer 5 ' ATTGAGCTCTCAGCGAACTCTCTCGAGGTATG3 '

PynC2 mrna length is 768bp

PynC3 gene: forward primer 5 ' ATAGAATTCATGGCGTCTCAGCTGCCCGCG3 '

Reverse primer 5 ' AGCAAGCTTTCACTTAGTTGGCGGGTACTTG3 '

PynC3 mrna length is 738bp

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 being undertaken synthesizing of " hundred make " production bacterium Cordyceps sinensis China pilose spore cDNA the first chain by following after total RNA, for follow-up each gene clone experiment.

Adopt synthetic cDNA the first chain of PrimeScript1st 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℃，5min。

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, carry out reverse transcription reaction by following condition.

42℃ 15～30min

70℃ 15min

Generalized case, there is a PolyA structure at eukaryote mRNA 3 ' end, 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, synthetic cDNA the first chain taking mRNA as template, the sequence (providing in PrimeScript1st 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, can obtain so cDNA first chain of all zymoprotein encoding genes in species by reverse transcription process.

Embodiment 9: " hundred make " produces the detection of clone, expression and the protein vigor of bacterium Cordyceps sinensis China pilose spore anabolism uridylic acid functional gene uridylic acid synthetic enzyme pynC gene

1, the pcr amplification of uridylic acid synthetic enzyme pynC1/C2/C3 gene

Taking cDNA the first chain of obtaining in embodiment 8 as template, pyn C1 gene primer with synthetic in embodiment 7: 5 ' AGA GAA TTC ATG TTG GCT GAC GCT AGG GTC3 ', 5 ' ATA AAG CTT TCA CCC TCT GCC GCA CCA ACT TG3 ', pyn C2 gene primer: 5 ' ATT GGA TCC ATG CAG TAC ACT GGC GGC TC3 ', 5 ' ATT GAG CTC TCA GCG AAC TCT CTC GAG GTA TG3 ', pyn C3 gene primer: 5 ' ATA GAA TTC ATG GCG TCT CAG CTG CCC GCG3 ', 5 ' AGCAAGCTTTCACTTAGTTGGCGGGTACTTG3 ' carries out Pfu archaeal dna polymerase pcr amplification reaction, condition setting is as follows:

Pfu pcr amplification reaction system:

Pfu DNA Ploymerase pcr amplification condition:

2, uridylic acid synthetic enzyme pynC1, pynC2, pynC3 gene PCR product gel electrophoresis detection

Concrete detection method is: 1) it is uniformly dissolved 0.9% the sepharose microwave-oven-heating preparing; 2) get 15mL gel, in the time that gel is cooled to 50 DEG C 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 Buffer1.5 μ L and ddH ₂it is 10 μ L that O4 μ L uses liquid-transfering gun loading, applied sample amount after mixing; 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 5V/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.

The size of transcribing group order-checking prediction uridylic acid synthetic enzyme pynC1, pynC2 and pynC3 gene is 414bp, 768bp, 738bp, and agarose gel electrophoresis result (Fig. 3) shows successfully to have amplified uridylic acid synthetic enzyme pynC1, pynC2 and pynC3 gene.

3, the base A that adds of uridylic acid synthetic enzyme pynC1, pynC2 and pynC3 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 DEG C add A base 20min, finally purify with AxyPrep PCR cleaning agents box.

4, being connected of uridylic acid synthetic enzyme pynC1, pynC2 and pynC3 gene and cloning vector

Cloning vector pMD18-T Vector is purchased from TaKaRa company (TaKaRa code D101A), its physical map is shown in Fig. 4, uridylic acid synthetic enzyme pynC1, pynC2 are connected respectively to construction recombination plasmid pMD18-T/pynC1, pMD18-T/pynC2 and pMD18-T/pynC3 with pynC3 gene with cloning vector, physical map is shown in Fig. 5, and linked system and condition of contact are as follows.

Linked system:

Condition of contact: 16 DEG C, 16h; Deactivation: 65 DEG C, 15min.

5, the conversion of uridylic acid synthetic enzyme recombinant plasmid pMD18-T/pynC1, pMD18-T/pynC2, pMD18-T/pynC3

Respectively recombinant plasmid pMD18-T/pynC1, pMD18-T/pynC2, pMD18-T/pynC3 are proceeded in intestinal bacteria E.coli JM109 and to build recombinant bacterium E.coliJM109/pMD18-T/pynC1, the E.coliJM109/pMD18-T/pynC2, the E.coliJM109/pMD18-T/pynC3 that carry uridylic acid synthetic enzyme pynC1, pynC2 and pynC3 gene.Concrete steps are: 1) 10 μ L reaction systems are gone in competent cell E.coliJM109 to ice bath 30min; 2) thermal shock: 42 DEG C, 90s; 3) ice bath: 2-3min; 4) add 800 μ L liquid LB, 37 DEG C, 250rpm, 1h; 5) spread plate (containing Amp resistance); 6) 37 DEG C of incubator overnight incubation.

6, the screening of uridylic acid synthetic enzyme E.coli JM109/pMD18-T/pynC1, E.coli JM109/pMD18-T/pynC2, the positive recombinant bacterium of E.coli JM109/pMD18-T/pynC3

Bacterium colony PCR can extract genomic dna, and directly carries out pcr amplification taking the DNA that exposes after thalline pyrolysis as template, and whether the method is easy and simple to handle, quick, can Rapid identification bacterium colony be the positive bacterium colony that contains object plasmid.First bacterium colony, adds and contains in the 1.5mL centrifuge tube of 50 μ L sterilized waters with toothpick picking list bacterium colony, and boiling water bath 30min is then centrifugal using 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 uridylic acid synthetic enzyme recombinant plasmid pMD18-T/pynC1, pMD18-T/pynC2, pMD18-T/pynC3

After the positive recombinant bacterium liquid LB culture medium culturing that bacterium colony PCR is detected 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, SEQ ID No.4 and SEQ ID No.6 have recombinated respectively to recombinant plasmid pMD18-T/pynC1, pMD18-T/pynC2 and pMD18-T/pynC3.

8, the structure of uridylic acid synthetic enzyme recombinant expression plasmid pET-28a/pynC1, pET-28a/pynC2, pET-28a/pynC3

Experiment is the principle at expression in escherichia coli according to foreign gene, and expression vector pET-28a and uridylic acid synthetic enzyme pynC1, pynC2, pynC3 gene restriction enzyme site comparison situation, determine that pynC1 and pynC3 are EcoR I and Hind III double enzyme site, pynC2 is BamHI/SacI restriction enzyme site, and recombination bacillus coli E.coli JM109/pMD18-T/pynC1, E.coli JM109/pMD18-T/pynC2 and E.coli JM109/pMD18-T/pynC3 are carried out to the cultivation of liquid LB test tube shaker, recombinant plasmid extraction.

The recombinant plasmid pMD18-T/pynC1 of uridylic acid synthetic enzyme pynC1 and pynC3 gene and pMD18-T/pynC3 and expression vector pET-28a respectively with BamHI/SacI III restriction enzyme 37 DEG C respectively enzyme cut process 6h, the recombinant plasmid pMD18-T/pynC2 of pynC2 gene and expression vector pET-28a process 6h with BamHI/SacI restriction enzyme at 37 DEG C, and it is as follows that enzyme is cut system:

BamH I/SacI double digestion system:

Enzyme is cut and is finished rear 65 DEG C of deactivation 15min, then respectively with Axygen DNA gel recovery test kit reclaim, purifying.

Uridylic acid synthetic enzyme pynC1, pynC2, pynC3 gene and expression vector pET-28a spend the night with 16 DEG C of connections of T4 ligase enzyme after double digestion, purifying again, build recombinant expression plasmid pET-28a/pynC1, pET-28a/pynC2, pET-28a/pynC3, its building process is shown in Fig. 6, builds recombinant expression plasmid pET-28a/pynC1, the pET-28a/pynC2, the pET-28a/pynC3 collection of illustrative plates that obtain and sees Fig. 7.Linked system is composed as follows:

Linked system:

9, uridylic acid synthetic enzyme recombinant expression plasmid pET-28a/pynC1, the conversion of pET-28a/pynC2, pET-28a/pynC3 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 (50mg/L) kantlex (Kan) resistance 37 DEG C 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 uridylic acid synthetic enzyme recombinant bacterium E.coli BL21/pET-28a/pynC1, E.coli BL21/pET-28a/pynC2 and E.coli BL21/pET-28a/pynC3

Be inoculated in the LB liquid nutrient medium that 5mL contains Kan resistance (50mg/L) 37 DEG C, 250r/min overnight incubation by being accredited as positive mono-clonal.Get 1mL culture, transferred in the LB liquid nutrient medium of the fresh Kan of the containing resistance of 50mL (50mg/L) 37 DEG C, 250r/min are cultured to cell concentration OD600 and are about 0.6～0.8 left and right.To the IPTG inducing culture 8h that adds respectively finite concentration (240mg/mL) in culture.Collecting wet thallus surveys for electrophoretic analysis and enzyme biopsy.

11, uridylic acid synthetic enzyme recombinant bacterium E.coli BL21/pET-28a/pynC1, E.coli BL21/pET-28a/pynC2 and E.coli BL21/pET-28a/pynC3 expression product SDS-PAGE analyze

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 8h, 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, " C1 " in Fig. 8, " C2 ", " C3 " swimming lane is respectively recombinant bacterium E.coli BL21/pET-28a/pynC1, E.coli BL21/pET-28a/pynC2, the uridylic acid synthetase albumen pynC1 (being shown in SEQ ID No.1 through its aminoacid sequence of sequence verification) that E.coli BL21/pET-28a/pynC3 expresses, pynC2 (being shown in SEQ ID No.3 through its aminoacid sequence of sequence verification), the SDS-PAGE figure of pynC3 (being shown in SEQ ID No.5 through its aminoacid sequence of sequence verification).

12, the protein-active of uridylic acid synthetic enzyme recombinant bacterium E.coli BL21/pET-28a/pynC1, E.coli BL21/pET-28a/pynC2 and E.coli BL21/pET-28a/pynC3 detects

Enzyme liquid preparation: take recombinant bacterium E.coli BL21/pET-28a/pynC1, E.coli BL21/pET-28a/pynC2 and the each 0.5g of E.coli BL21/pET-28a/pynC3 wet thallus (dry weight 0.1g) that method 10 is collected, use respectively phosphate buffered saline buffer (50mM, pH8.0) 15mL to suspend, after ultrasonication (power 350W, broken 2s, interval 2s, common ultrasonication 5min), be used as catalysis enzyme.

Uridylic acid synthetic enzyme pynC transformation system: add respectively E.coli BL21/pET-28a/pynC1, E.coli BL21/pET-28a/pynC2 and E.coli BL21/pET-28a/pynC3 ultrasonication thalline mixed solution 10mL, 0.1g orotidylic acid in 50mL conversion bottle, 30 DEG C, 150r/min conversion reaction 2～3h, after conversion finishes, conversion fluid is centrifugal, get respectively supernatant standby with subsequent detection.

Detection method: the conversion situation of high performance liquid chromatography detection by quantitative uridylic acid.Pre-treatment: after conversion fluid is centrifugal, supernatant liquor is crossed 0.22 μ m microporous membrane and detected for high performance liquid chromatography.Condition: chromatographic column: (4.6mm × 250mm i.d.5 μ m) for Agilent C18 post; Column temperature: 35 DEG C; Sampling volume: 20 μ L; Flow velocity 1mL/min; Detect wavelength 260nm; Moving phase: A: ultrapure water; B: methyl alcohol.Gradient elution: 0min, 15%B, keeps 3min; 3.0-3.5min, 15-24%B; 3.5min, 24% keeps 5min; 8.5-9.0min, 24-35%B; 35%B keeps 6min; 15.0-16.0min, 35-85%B; 85%B keeps 6min; 22.0-22.5min, 85%-15%B; 15%B keeps 5min.

The making of typical curve: accurately take uridylic acid standard substance, about 1.00mg-2.00mg, ultrapure water constant volume.Standard substance 6 gradients of preparation (1 μ g/mL, 30 μ g/mL, 60 μ g/mL, 90 μ g/mL, 120 μ g/mL, 150 μ g/mL).Then loading successively, each concentration repeats for 5 times.After the reference liquid of getting certain volume maximum concentration mixes, constant volume, then HPLC analyzes the optimal conditions that uridylic acid detects.

Detect and calculate through above-mentioned chromatographic condition, we draw to draw a conclusion: the expressed oxidasic high specific enzyme of uridylic acid synthetic enzyme (Specific Activity) alive of uridylic acid synthetic enzyme recombinant bacterium E.coli BL21/pET-28a/pynC1, E.coli BL21/pET-28a/pynC2 and E.coli BL21/pET-28a/pynC3 is respectively 65mol/min/mg, 63mol/min/mg, 59mol/min/mg, and substrate conversion efficiency is respectively 76%, 72% and 69%.

Claims (8)

1. from a uridylic acid synthetic enzyme for Cordyceps sinensis China pilose spore, the aminoacid sequence that it is characterized in that described enzyme is shown in SEQ ID No.3.

2. prepare the application in uridylic acid from the uridylic acid synthetic enzyme of Cordyceps sinensis China pilose spore at biocatalysis orotidylic acid as claimed in claim 1.

3. application as claimed in claim 2, it is characterized in that described being applied as: the broken mixed solution taking the wet thallus containing the acquisition of Cordyceps sinensis China pilose spore uridylic acid synthetic enzyme thalline fermentation culture after cytoclasis is as catalyzer, taking orotidylic acid as substrate, in the transformation system that the buffered soln that is 6.5～8.5 in pH forms, conversion reaction 2～3h under 30 DEG C, 150rpm condition, after reaction finishes, by reacting liquid filtering, get filtrate and be the crude product containing uridylic acid, described crude product separation and purification obtains uridylic acid.

4. application as claimed in claim 3, the starting point concentration that it is characterized in that described substrate is 10g/L, and the volumetric usage of described catalyzer is 100mL/g substrate, and the dry mycelium concentration of described catalyzer is 6.7mg/mL.

5. the gene of enzyme described in the claim 1 of encoding.

6. gene as claimed in claim 5, is characterized in that the nucleotides sequence of described gene is classified SEQ ID No.4 as.

As described in claim 5 or 6 gene in the application building in the genetic engineering bacterium of can biocatalysis orotidylic acid preparing uridylic acid.

8. application as claimed in claim 7, it is characterized in that described being applied as: build the recombinant vectors that contains described Cordyceps sinensis China pilose spore uridylic acid synthase gene, described recombinant vectors is converted in intestinal bacteria, the recombination engineering bacteria obtaining carries out inducing culture, and nutrient solution separation and purification obtains the somatic cells that contains Cordyceps sinensis China pilose spore uridylic acid synthetic enzyme.