CN109385410A

CN109385410A - Bite the RNA polymerase termination sequence and its identification method and application of Jun body Syn5 in ocean

Info

Publication number: CN109385410A
Application number: CN201811067462.XA
Authority: CN
Inventors: 朱斌; 吴慧; 夏恒; 陆雪玲; 黄锋涛; 余兵兵; 闫艳; 成锐
Original assignee: Huazhong University of Science and Technology; Shenzhen Huazhong University of Science and Technology Research Institute
Current assignee: Huazhong University of Science and Technology; Shenzhen Huazhong University of Science and Technology Research Institute
Priority date: 2018-09-13
Filing date: 2018-09-13
Publication date: 2019-02-26

Abstract

The invention discloses RNA polymerase termination sequences and its identification method and application that Jun body Syn5 is bitten in ocean, belong to microbial nucleic acids metabolic enzyme technical field.The termination sequence contains at least one ATCTGT sequence units.Identification method is to be inserted into foreign gene at the multiple cloning sites of carrier, and the promoter sequence in the carrier is replaced with Syn5 promoter sequence, obtains recombinant vector；The recombinant vector is transcribed in vitro, it recycles the RNA product other than the RNA of overall length transcription and carries out 3 ' RACE sequencing, corresponding termination sequence contains at least one ATCTGT sequence units and contains at least one ATCTGT sequence units to get to bacteriophage Syn5RNA polymerase transcription termination sequence.The transcription terminator can be applied to RNA synthetic biology.The present invention identifies single subunit RNA polymerase transcription terminator of marine bacteriophage Syn5, compensates for currently to the deficiency of Syn5RNA polymerase transcription characteristic research, identifies the current most simplified sequence units for causing polymerase that termination is transcribed in vitro.

Description

Bite the RNA polymerase termination sequence and its identification method and application of Jun body Syn5 in ocean

Technical field

The invention belongs to microbial nucleic acids metabolic enzyme technical fields, and in particular to bite the RNA polymerase of Jun body Syn5 in ocean Terminator and its identification method and application.

Background technique

RNA (ribonucleic acid) polymerase be using RNA or DNA (DNA) be template using nucleoside triphosphate as Substrate synthesizes the enzyme of RNA, is the synthetics of RNA.RNA is the carrier of hereditary information transmitting, is widely present in eucaryote, original In core biology, fractionated viral and viroid, have the function of numerous species and.Except previous well known three categories RNA:mRNA, rRNA And outside tRNA, Recent study find some novel RNA such as microRNA (miRNA) (Mohr and Mott., 2015), long non-coding RNA(lncRNA)(Dey et al.,2014)、Circular RNAs(circRNA)(Memczak Et al., 2013) etc. key effect is all played in different physiological processes.With deepening continuously for RNA correlative study, RNA Application value in terms of disease treatment is also increasingly shown.Be delivered to after synthesizing in vitro by design mRNA sequence intracellular It can be translated as any target protein, and mRNA translation is quickly, automatic degradation, the advantage compared to DNA can existed in vivo In will not be integrated into host genome, and work faster.In addition to being used as vaccine, mRNA drug also can be used as pharmaceutical grade protein supplement Or alternative medicine, treat other a variety of diseases.Currently, the technologies such as mRNA modification and preparation also achieve quantum jump, solve The stability and delivery problems of mRNA, this makes mRNA drug become a kind of ideal medicament forms, mRNA pharmaceutical industry Also increasingly mature, the drugmakers such as Moderna, BioNTech and CureVac have all formed mRNA drug products line abundant.It removes Outside mRNA, RNA interference (RNAi) is carried out to target gene using double-stranded RNA and utilizes the gene editing of gRNA guidance (CRISPR gRNA) technology etc. all has preferable prospect in disease treatment.

The development of RNA correlative study and application proposes very high challenge to RNA external preparation, and traditional chemical synthesis side Method is only limitted to tens nucleotide length below of synthesis, also steeply rises when length increases cost.However it is designed to encode The mRNA of protein generally reaches thousands of a nucleotide, it is clear that this method is no longer applicable in, and can only carry out body by RNA polymerase Outer transcription is to synthesize RNA.Although RNA polymerase is many kinds of, the RNA polymerase of most of organisms all has complicated Asia Base composition and regulatory mechanism, are not suitable for use in the external synthesis machine of RNA.1970s scientists find that one kind comes from Single subunit RNA polymerase of Escherichia coli T7 bacteriophage, structure simply have specific transcription (identifying special promoter) The features such as high with transcriptional efficiency, 1984 by a time cloning after be just widely used in external synthesis, the expression of recombinant protein of RNA (the high expression system of bacterium) etc. (Davanloo et al., 1984) becomes the unique method of external synthesis long-chain RNA, in biology Chemical research and RNA synthesis field have a wide range of applications.The bacteriophage T3RNA polymerase and SP6 RNA of subsequent discovery are poly- Synthase is all relatively close with T7 RNA polymerase affiliation and has similar property, with the continuous hair of the external synthesis application of RNA Exhibition and deeply, this kind of list subunit RNA polymerase all shows many very important disadvantages, including product end is inhomogenous, right Starting base has stronger Preference, incorporation modified nucleoside acid low efficiency, and transcription can generate many interruptions rich in higher structure RNA Product etc. (Lyakhov et al., 1998；Chamberlin and Ring, 1973), cause to synthesize in many cases RNA is unable to satisfy the requirement of follow-up study and application.

Although scientists are subsequent to have carried out many transformations around T7 RNA polymerase, there is no obtain for its major defect To improvement, it is increasingly becoming the factor of limitation this popular domain research and development of RNA.Facts proved that due to early stage identification Bacteriophage type is limited, this fermentoid is not the external synthetics enzyme of optimal RNA.With molecular biology research in recent years With flourishing for sequencing technologies, discloses marine microorganism quantity and type is very huge, more and more novel bacteriophages It is found, wherein many novel enzyme instruments may be contained.One of the main inventive people of this patent doctor Zhu Bin is in Harvard University Medical College studies marine bacteriophage Syn5 during working, and host is the most cyanobacteria of ocean mileage amount. By killing in ocean 20% biomass, bacteriophage Syn5 plays a significant role in maintaining marine environment energy balance (Pope et al.,2007).By with t7 rna polymerase gene carry out similitude compare, protein purification and characterization analysis The methods of, doctor Zhu Bin identifies completely new single subunit RNA polymerase (the Zhu et from marine bacteriophage Syn5 al.,2013).It is compared to T7 RNA polymerase, this enzyme from ocean can be dense in a larger range of temperature and salt The synthesis of RNA is catalyzed under the conditions of degree, transcription extension ability also greatly improves, also no very stringent to base is originated after promoter Limitation.The biggest advantage is to can carry out accurate overall length transcription and keep the uniform of product end for Syn5 RNA polymerase Property (Zhu et al., 2014；Zhu et al.,2015；US patent WO2015024017).Syn5 RNA polymerase is captured The bottleneck that T7 RNA polymerase is encountered in biosynthesis field, Harvard University has been Syn5 RNA polymerase application Liang Xiang state Border patent.Currently, Syn5 RNA polymerase shows very big prospect in synthetic biology application.

Although Syn5 RNA polymerase transcriptional promoter sequence and transcription extension property etc. be studied it is fully aware of, But its tanscription termination characteristic is also not well known.Tanscription termination is also re-recording system as extending with transcription initiation with transcription Important component, it is particularly significant to the yield and length of control transcription product in the application.In the feelings for not knowing transcription terminator Under condition, some negative effects may be generated to the external synthesis application of Syn5 RNA polymerase.In protein expression system, Bu Nengyou The tanscription termination of effect not only can largely reduce outside the transcriptional efficiency of target gene, also will affect the expression water of downstream components It is flat, such as antibiotics resistance gene, plasmid copy number control element or the inhibition albumen for reducing background expression level (Mairhofer et al.,2015).So far, still not related to the termination performance of the RNA polymerase of bacteriophage Syn5 Research report.

Summary of the invention

The present invention is directed in the prior art there has been no the termination sequence of the RNA polymerase of bacteriophage Syn5, provides phagocytosis Body Syn5 RNA polymerase transcription terminator and its identification method and application.

It is according to the invention in a first aspect, provide a kind of marine bacteriophage Syn5 RNA polymerase transcription terminator, The termination sequence contains at least one ATCTGT sequence units.

It is another aspect of this invention to provide that providing the marine bacteriophage Syn5 RNA polymerase transcription terminator Identification method contains following steps:

(1) foreign gene is inserted at the multiple cloning sites of carrier, and the promoter sequence in the carrier is replaced with Marine bacteriophage Syn5 promoter sequence obtains recombinant vector；The recombinant vector is subjected to digestion using restriction enzyme, The recombinant vector linearized；

(2) the outer responsive transcription system of construct, the in-vitro transcription reaction system is with the recombination of step (1) linearisation Carrier is as template, using bacteriophage Syn5 RNA polymerase as RNA polymerase, with tetra- kinds of nucleosides three of ATP, GTP, CTP and UTP Phosphoric acid makes the outer responsive transcription of the recombinant vector generating body of the linearisation as raw material；DNA enzymatic is added to eliminate as template The recombinant vector of linearisation, the RNA product that then purifying transcription obtains；

(3) the RNA product for obtaining step (2) carries out gel electrophoresis, the RNA product other than the RNA of recycling overall length transcription And 3 ' RACE sequencing is carried out, the corresponding termination site of RNA product other than the RNA of the overall length transcription contains at least one ATCTGT sequence units contain at least one ATCTGT sequence to get to marine bacteriophage Syn5 RNA polymerase transcription terminator Column unit.

Preferably, step (1) carrier is pET28b, and step (1) restriction enzyme is restriction enzyme BglII。

Preferably, step (2) time that reaction is transcribed in vitro is 1h-2h.

Preferably, the concentration in responsive transcription system is 1 μ to step (2) the bacteriophage Syn5 RNA polymerase in vitro Μ-2μΜ。

Preferably, the concentration in responsive transcription system is 20ng/ to the recombinant vector of step (2) described linearisation in vitro ul-40ng/ul。

It preferably, further include RNase inhibitor in step (2) the in-vitro transcription reaction system.

It preferably, further include Tris-HCl buffer, MgCl in step (2) the in-vitro transcription reaction system₂, sub- essence Amine, dithiothreitol (DTT) and pyrophosphatase.

Exist it is another aspect of this invention to provide that providing the marine bacteriophage Syn5 RNA polymerase transcription terminator Application in RNA synthetic biology.

Preferably, the application is that the bacteriophage Syn5 RNA polymerase transcription terminator is inserted into transcription templates It is in transcription templates by the bacteriophage Syn5 RNA polymerase transcription terminator to terminate responsive transcription or the application Same sense mutation is carried out to continue responsive transcription.

In general, through the invention it is contemplated above technical scheme is compared with the prior art, mainly have below The utility model has the advantages that

(1) present invention express and has been purified to single subunit RNA polymerase from marine bacteriophage Syn5, it is therefore an objective to Its transcription terminator is identified, efficient transcription terminator element is obtained.The present invention identifies marine bacteriophage Syn5's Single subunit RNA polymerase transcription terminator is compensated for currently to the deficiency of Syn5 RNA polymerase transcription properties research, is identified One current most simplified sequence units for causing polymerase that termination is transcribed in vitro.

It (2), not only can be high during the Syn5 RNA polymerase transcription terminator of identification being applied to in-vitro transcription The termination for precisely transcribing of effect；And on the other hand, the bacteriophage Syn5 RNA polymerase is turned in transcription templates It records termination sequence and carries out same sense mutation to continue responsive transcription.

(3) Syn5 RNA polymerase transcription terminator of the present invention is sequence-specific terminator, rather than is structure The terminator of dependent form, therefore can be neatly using the continuation for terminating either responsive transcription for transcription.

Detailed description of the invention

Fig. 1 show the purification effect figure of SDS-PAGE electrophoresis detection Syn5 RNA polymerase in embodiment 1, wherein M generation Table albumen Marker.

Fig. 2, which is shown, to be transcribed in vitro using T7 RNA polymerase and Syn5 RNA polymerase in embodiment 2 Terminate result figure；The top is the RNA product of overall length transcription, and lower section is the RNA product terminated in advance.

Fig. 3 (a) is that Syn5 RNA polymerase transcription terminator sequences are applied to the result in being transcribed in vitro in embodiment 3 Figure；The transcription termination efficiency of swimming lane 2- swimming lane 6 in corresponding diagram 3 (a) shown in Fig. 3 (b).

Specific embodiment

In order to make the objectives, technical solutions, and advantages of the present invention clearer, with reference to the accompanying drawings and embodiments, right The present invention is further elaborated.It should be appreciated that described herein, specific examples are only used to explain the present invention, not For limiting the present invention.As long as in addition, technical characteristic involved in the various embodiments of the present invention described below that Not constituting conflict between this can be combined with each other.In the situation for not departing from the present invention above-mentioned technical idea and disclosed spirit Under, according to ordinary skill knowledge and customary means, various equivalent, replacements, modification or change are made, the present invention is both fallen within The range of protection.

The amplification of embodiment 1:Syn5 rna polymerase gene and protein expression and purification

(1) Syn5 rna polymerase gene and protein expression are expanded

PCR amplification obtains Syn5 rna polymerase gene, and gene order is cloned into the enzyme of prokaryotic expression carrier pET-24a Between enzyme site NdeI and NotI, in the DNA fragmentation of 33 bases longs of gene 5 ' terminal fusion, described in DNA fragmentation coding Company flexible is kept between histidine tag and label and Syn5 RNA polymerase as shown in SEQ ID NO:1 in sequence table Peptide fragment is connect, joining peptide sequence is Gly-Gly-Gly-Gly-Gly.Syn5 rna polymerase gene sequence is shown according to existing research Column region contains the specific promoter sequence of its transcription identification, thus base sequence progress same sense mutation (is not changed coding Amino acid), the gene order in carrier pET-24a is finally inserted into as shown in SEQ ID NO:2 in sequence table, the weight that will be built Group carrier Transformed E .coli BL21 (DE3) express bacterial strain, choose bacterium expand culture, by bacterium be placed in containing 50ug/ml card that LB train 37 DEG C of shaking table cultures are supported in base to OD₆₀₀The isopropyl-beta D-thio galactopyranose of 0.5mM final concentration is added close to 1.2 in value Glycosides (IPTG) is in 28 DEG C of shaking table inducing expression 3h.

(2) Syn5 RNA polymerase is purified

Bacteria lysis: after IPTG inducible protein expression 3h is added, 4 DEG C, 5000rpm is centrifuged 20min, removes supernatant and collects bacterium Body precipitating, thallus is sufficiently resuspended in containing 20mM Tris-HCl (pH7.5), 2M Nacl, 0.5mg/ml lysozyme, 0.5mM In the lysate of DTT, protein lysate is then frozen in -80 DEG C, to be solidified be placed on melts 1h on ice, then be placed in - 80 DEG C of multigelations are twice.Protein liquid is placed in high-speed refrigerated centrifuge 4 DEG C, 35000rpm centrifugation 1h, after centrifugation in separation Clearly, the supernatant isolated is passed through to the membrane filtration in 0.45 μm of aperture, further to remove impurity, filtered protein liquid can Carry out subsequent ni-sepharose purification or 4 DEG C of of short duration preservations.

Ni-sepharose purification: prepare containing 20mM Tris-HCl (pH7.5), 2M NaCl elution buffer, separately configure 20mM, The imidazole solution of tri- kinds of concentration of 50mM, 100mM is spare.Nickel column is balanced using the elution buffer of 10 times of nickel column packing volumes, so Afterwards will before filtered protein lysate be added in nickel column, flow out the liquid in nickel column by gravity, in next step according to Column is crossed by the imidazole solution that the sequence of low concentration to high concentration is gradually added 20mM, 50mM, 100mM, elutes non-specific binding Foreign protein and final eluting the protein competition in conjunction with nickel column.The protein liquid that each concentration elutes needs to use Several clean cryopreservation tubes save, and finally detect all protein liquids eluted by SDS-PAGE electrophoresis, select The Syn5 RNA polymerase of higher degree is saved in 4 DEG C.

Phosphocellurose column chromatography: albumen is added in phosphocellurose column, is prepared and is contained 20mM potassium dihydrogen phosphate (pH7.5), 1mM DTT, 1mM EDTA, 10%glycerol, 20mM KCl start buffer and contain 20mM di(2-ethylhexyl)phosphate Hydrogen potassium (pH7.5), 1mM DTT, 1mM EDTA, 10%glycerol, the elution buffer of 1M KCl are spare, use start buffer 5-10 column volume is balanced, salt ionic concentration (by 0.02M KCl to 1M KCl) progress is continuously improved in a manner of continuously eluting All liquid eluted are carried out Coomassie brilliant blue detection by elution, and the protein liquid that color becomes blue passes through SDS-PAGE electrophoresis It is detected, Syn5 RNA polymerase about is eluted out in the high salt concentration of 700mM, collects albumen.

Gel permeation chromatography (molecular sieve): by albumen obtained in the previous step with super filter tube (Millipore) be concentrated to 1ml with It is interior, then using 200ml gel filtration prepacked column Superdex 200 carry out gel permeation chromatography, prepare in advance containing The elution buffer of 20mM Tris-HCl (pH 7.5), 2M NaCl, 0.5mM DTT, 0.5mM EDTA are balanced with the buffer After 2 column volume of molecular sieve, albumen is added in column, starts to be eluted with eluent after protein liquid fully enters filler, The flow control eluted using peristaltic pump collects elution albumen with automatic collector, by all liquid eluted in 1ml/min Body carries out Coomassie brilliant blue detection, and the protein liquid that color becomes blue is detected by SDS-PAGE electrophoresis, the purity collected Higher protein liquid.

Albumen dialysis: the protein liquid that gel permeation chromatography is obtained is dialysed after appropriate volume is concentrated by ultrafiltration, Then plastics clamp opening is added in bag filter in albumen, bag filter is placed in 1L and contains 100mM NaCl, 50mM Tris-HCl (pH 8.0), 0.1mM DTT, 0.1mM EDTA, 50% glycerol dialyzate in, be placed on magnetic stirring apparatus using magnetic bead stir Promote solution exchange, dialysis 3h or more replaces clean dialyzate, then proceedes to dialysis 3h or more, and finally replacement dialyzate is saturating again Overnight, albumen is placed in -20 DEG C of preservations after collecting dialysis for analysis.

(3) SDS-PAGE electrophoresis detection Syn5 RNA polymerase purification effect

SDS-PAGE electrophoresis is carried out after dialysis, then using coomassie brilliant blue staining to the Syn5 RNA polymerase after dialysis Purity detected, as a result as shown in Figure 1, it can be seen that protein band is relatively simple after dialysis, illustrate Syn5 RNA polymerize The purity that enzyme obtains after the above purification step is higher.

Embodiment 2: it is transcribed in vitro using T7 RNA polymerase and Syn5 RNA polymerase

(1) acquisition and purifying of responsive transcription template

PCR amplification NrsPOL gene is cloned into the length for increasing carrier in the multiple cloning sites of carrier pET28b, obtains After recombinant vector, the T7 promoter sequence in carrier is substituted for Syn5 promoter sequence, is finally built into two containing difference Then the recombinant vector of transcripting promoter is linearized using the restriction enzyme BglII before promoter, use Purification kit DNA Clean&Concentrator^TM- 5 (being purchased from ZYMO RESEARCH biotechnology company) are to linearisation DNA Product is purified.

(2) reaction is transcribed in vitro

Except RNA polymerase and transcription templates, other ingredients are consistent in in-vitro transcription reaction system, including 40mM Tris- HCl (pH7.9), 6mM MgCl₂, 2mM spermidine, 10mM DTT, 200 μ Μ ATP, GTP, CTP, UTP, the inhibition of 0.3ul RNA enzyme Agent, 0.2ul pyrophosphatase mend DEPC water to 10ul.Template containing T7 promoter and Syn5 promoter is separately added into final concentration 1 μ Μ t7 rna polymerase and 2 μ Μ Syn5 RNA polymerases linearize the final concentration of 20ng/ul of transcription templates.By T7 transcription System and Syn5 transcription system are respectively placed in 37 DEG C and 30 DEG C reaction 2h.2ul RQ1RNase- is then added in the reaction system Free DNase (Promega) eliminates linearisation template, finally uses RNA Clean&Concentrator in 37 DEG C of reaction 1h^TM- 5 (ZYMO RESEARCH) purifying RNA products.It configures 2% agarose gel and electrophoresis is carried out to RNA product, dyed using EB, as a result As shown in Figure 2.As shown in Figure 2, the RNA production that transcription is terminated except acquisition in T7 transcription terminator is carried out using t7 rna polymerase Beyond the region of objective existence, there is also apparent overall length transcription products.Transcribed to obtain the site terminated at two, position using Syn5RNA polymerase After T7 termination sequence on pET28b carrier, Syn5 RNA polymerase is not sent out at the T7 transcription terminator with loop-stem structure Raw tanscription termination, in addition equally exists the RNA of part overall length transcription.Syn5 RNA polymerase is generated on pET28b carrier Termination site T1 and T2 carry out 3 ' RACE sequencing, the sequence such as SEQ ID NO:3-7 institute that termination site T1 is sequenced at two Show, for the sequence that termination site T2 is sequenced as shown in SEQ ID NO:8-12, finally found that through sequence alignment leads to Syn5 The key core sequence that RNA polymerase terminates is ATCTGT.

Embodiment 3: Syn5 RNA polymerase transcription terminator is applied to be transcribed in vitro

(1) acquisition and purifying of responsive transcription template

Syn5 rna polymerase promoter sequence is inserted at 422 position of pUC19 carrier sequence, obtains recombinant vector Afterwards, it is in addition inserted into CATCTGTTTTTTTT, CATCTGTTTTTTTT respectively at 1500 position of sequence on the basis of the carrier (existing research behind terminator the result shows that add for X2, CATCTGTTTTTTTT X3, ATCTGT X3, ATCTGT X6 base sequence T is improved effect to efficiency is terminated), 6 kinds of recombinant vectors are obtained, line is carried out to plasmid template using restriction enzyme NdeI Property, linearize product purification kit DNA Clean&Concentrator^TM- 5 (ZYMO RESEARCH) are purified.

(2) reaction is transcribed in vitro

The system of reaction is transcribed in vitro are as follows: 40mM Tris-HCl (pH7.9), 6mM MgCl₂, 2mM spermidine, 10mM DTT, 200 μ Μ ATP, GTP, CTP, UTP, 0.3ul RNase inhibitor, 0.2ul pyrophosphatase, 2 μ Μ Syn5 RNA polymerization Enzyme, 20ng/ul transcription templates mend DEPC water to 10ul.Reaction system is mixed takes 1ul to transcribe after being placed on 30 DEG C of transcription 2h The laggard row agarose gel electrophoresis detection of sample-loading buffer of formamide and EDTA configuration is added in product, as a result as shown in figure 3, making It is imitated with VisionWorks software statistics band gray value and according to the termination that each swimming lane reaction is calculated in RNA primer size Rate.Fig. 3 (a) is that Syn5 RNA polymerase transcription terminator sequences are applied to the result figure in being transcribed in vitro in embodiment 3；Swimming Road 1 is the pUC19 carrier transcription result for being only inserted into Syn5 rna polymerase promoter sequence, and swimming lane 2 to swimming lane 6 is respectively to carry Be additionally inserted at 1500 base of body CATCTGTTTTTTTT X1, CATCTGTTTTTTTT X2, CATCTGTTTTTTTT X3, The transcription result that ATCTGT X3, ATCTGT X6 base sequence obtain；The top band is DNA template, and lower section band is RNA production Object；The transcription termination efficiency of swimming lane 2- swimming lane 6 in corresponding diagram 3 (a) shown in Fig. 3 (b).By Fig. 3 it is known that swimming lane 6 is being inserted into When 36 base sequences of ATCTGT X6, Syn5 RNA polymerase transcription termination efficiency highest has reached nearly 95% termination effect Rate terminates efficiency and significantly improves, illustrate only to pass through raising compared to the CATCTGTTTTTTTT X3 and other base sequences of insertion The number of repetition of insertion ATCTGT can further increase termination efficiency in the case where introducing less base quantity.

<110>Central China University of Science and Technology

Huazhong Tech Univ Inst., Shenzhen

<120>the RNA polymerase termination sequence and its identification method and application of Jun body Syn5 is bitten in ocean

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gcagttgatg ccgccgaggt ctacaagcat ctgaagaacg ccgatcacca tcatctggcg 300

ctcatcacga tgaagacagc cctggacgtc ctgggcaaag atcccgagcc acagatccaa 360

cagctgacca cagccattgg ccgcaacatc cagctggagc tccgcctcac gtactacgcc 420

gaggaaaacc cggagctcta caaacaggcc tcccgcttct tccacgcagg cactggcacc 480

cgccagaaag ccacggtgat caaactcaag ttcaaccgcg agggcattga gtgggaccaa 540

tggtcccgcg tcacctgtca caaggttggc caatggctca tgttggctat ggccgacgtc 600

accggctgga ttgaacgggc aaccgaccga accagtggag gacgcaaaac caagacccgc 660

atctgctact cccgcgagtt cttgcagcat cgggacacaa tcctcgcagc agctgagcag 720

ttggccttct gccagtggcc catgctttgc cctcccattg agtggtccaa cgaccacaac 780

ggtgggtacc tgagcgaaca gatccggcgg gtcaatccgc tgattcgtaa aacgggtcca 840

ttgggcaccc gtaagcaagg agacataccc cttgcgatgc tcaacaacct gcagggtcag 900

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gcacagatca gtcagaagaa ctggcgcacg accgaggtca tgtatgtggc ccgcaagtac 1140

gccgacgagg cctccttctg gatgcccgcc agcttcgact atcgcggccg tgtttacttt 1200

ctgaacactg ccctcaaccc gcaggggact gacttcgaca aggcgctcct ttacttcgct 1260

gaggagggtc cggtcaacga atggtggcta tccttccacg tcgcgaccac ctacggcctc 1320

gacaaggaga ccatggtcaa ccgggtccaa tgggctcggg acaaccacga gctcatcgat 1380

cgcatcgcct ctgaccccgt ccgccatacc gagtggcacg acgctgacga gccctggtgc 1440

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ggcctcccta tcgggatcga cgccacctgt tcaggcctgc agcacctcgc tgctatgacc 1560

cgctgcggac gcacagccgc cctggtcaac gtgacaccga ccgacaagcc ggccgatgct 1620

tacaagaccg tggcgcaagc atccctcaag catctcccca aggagcagca cgagtggatc 1680

acccgcaagg tcaccaaaag gcccgtcatg tgcacaccct acggggtgac catgtcatcg 1740

gcccgcggct acatccgcga tcagctggtg aaggacggcc acaaggagga cctccgatct 1800

cctggcgtgc tcaacggcat cgtcaaggcg atctttaatg aggccatccc tgaggtcatc 1860

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ggtgattcca ccatcacgtg gaccacgccc tcaggcttcg aggttgttca agacctcaaa 1980

aagtccaaga cctacgaggt caagacccgc atcatgggcg gagcacggat caagctccaa 2040

gtgggcgacg ggttcaccga cgagcccgac cgtgaccacc acaaaagcgc actggctccc 2100

aacgtggtgc acagcaacga tgcgtctctc ctccacctga ccttcgcctt ctgggacaag 2160

cccttcacgg tcatccatga ctgtgtcctg ggccgttcct gcgacatgga tcagatgggc 2220

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gaccaggtgg gcgttgagct ccctgtcgac ctgatcaaaa acacgctcga catcgacagc 2340

gtcaaccagt ccctttactt cttctcctga 2370

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Claims

1. marine bacteriophage Syn5 RNA polymerase transcription terminator, which is characterized in that the termination sequence contains at least one A ATCTGT sequence units.

2. the identification method of marine bacteriophage Syn5 RNA polymerase transcription terminator as described in claim 1, feature exist In containing following steps:

(1) foreign gene is inserted at the multiple cloning sites of carrier, and the promoter sequence in the carrier is replaced with into ocean Bacteriophage Syn5 promoter sequence obtains recombinant vector；The recombinant vector is subjected to digestion using restriction enzyme, is obtained The recombinant vector of linearisation；

(2) the outer responsive transcription system of construct, the in-vitro transcription reaction system is with the recombinant vector of step (1) linearisation As template, using bacteriophage Syn5 RNA polymerase as RNA polymerase, with tetra- kinds of ribonucleoside triphosphotes of ATP, GTP, CTP and UTP As raw material, make the outer responsive transcription of the recombinant vector generating body of the linearisation；DNA enzymatic is added to eliminate as the linear of template The recombinant vector of change, the RNA product that then purifying transcription obtains；

(3) the RNA product for obtaining step (2) carries out gel electrophoresis, and the RNA product other than the RNA of recycling overall length transcription is gone forward side by side 3 ' RACE of row is sequenced, and the corresponding termination site of RNA product other than the RNA of the overall length transcription contains at least one ATCTGT Sequence units contain at least one ATCTGT sequence list to get to marine bacteriophage Syn5 RNA polymerase transcription terminator Member.

3. identification method as claimed in claim 2, which is characterized in that step (1) carrier is pET28b, step (1) institute Stating restriction enzyme is restriction enzyme BglII.

4. identification method as claimed in claim 2, which is characterized in that step (2) time that reaction is transcribed in vitro is 1h- 2h。

5. identification method as claimed in claim 2, which is characterized in that step (2) the bacteriophage Syn5 RNA polymerase exists The concentration being transcribed in vitro in reaction system is 1 μ Μ -2 μ Μ.

6. identification method as claimed in claim 2, which is characterized in that the recombinant vector of step (2) described linearisation is in vitro Concentration in responsive transcription system is 20ng/ul-40ng/ul.

7. identification method as claimed in claim 2, which is characterized in that also wrapped in step (2) the in-vitro transcription reaction system Include RNase inhibitor.

8. identification method as claimed in claim 2, which is characterized in that also wrapped in step (2) the in-vitro transcription reaction system Include Tris-HCl buffer, MgCl₂, spermidine, dithiothreitol (DTT) and pyrophosphatase.

9. marine bacteriophage Syn5 RNA polymerase transcription terminator as described in claim 1 is in RNA synthetic biology Using.

10. application as claimed in claim 9, which is characterized in that the application is to be inserted into the bacteriophage in transcription templates Syn5 RNA polymerase transcription terminator is in transcription templates by the bacteriophage to terminate responsive transcription or the application Syn5 RNA polymerase transcription terminator carries out same sense mutation to continue responsive transcription.