CN104651365A - Promoter of bovine alpha-actin gene and application thereof - Google Patents
Promoter of bovine alpha-actin gene and application thereof Download PDFInfo
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- CN104651365A CN104651365A CN201510063159.2A CN201510063159A CN104651365A CN 104651365 A CN104651365 A CN 104651365A CN 201510063159 A CN201510063159 A CN 201510063159A CN 104651365 A CN104651365 A CN 104651365A
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Abstract
The invention discloses a promoter of a bovine alpha-actin gene and an application thereof, belonging to the technical field of genetic engineering. The promoter is characterized in that the nucleic acid sequence of 170bp containing three SRE positive regulation elements inside the 262bp promoter segment of the bovine alpha-actin gene is subjected to PCR amplification by use of different primers to obtain two nucleic acid sequences of 170bp, and the two nucleic acid sequences of 170bp are connected in series with the upstream of the 262bp promoter segment (namely, 170bp-170bp-262bp) to build a promoter segment of alpha-actin, wherein the nucleotide sequence is shown by Seq ID No:1. The promoter has relatively high promoting activity and skeletal muscle specificity and can remarkably improve the expression activity of the alpha-actin gene in bovine skeletal muscle satellite cell and can be used as an efficient and specific skeletal muscle promoter and applied to the transgenic beef production for increasing the muscle output.
Description
Technical field
The present invention relates to promotor and the application thereof of an ox α-actin gene, belong to gene engineering field.
Background technology
Eukaryotic gene expression regulation is a complexity and tight process.In the research of transgenic animal, obtain and can the efficient specific promotor of widespread use the expression tool of foreign gene be of great significance.Promotor is an integral part of gene (gene), and controlling gene expresses the initial time of (transcribing) and the degree of expression.Promotor (Promoters), just as " switch ", determines the activity of gene.Since gene is into the Nucleotide (nucleotides) of sequence, so promotor also should be made up of DNA.Promotor not controlling gene itself is movable, but by be called this protein (proteins) of transcribing (transcription) factor to combine and controlling gene activity.Transcription factor, just as one side " flag ", commands the activity of enzyme (enzymes) (RNA polymerase polymerases).This enzyme manufactures the rna replicon of gene originally.Generally be divided into the various ways such as wide spectrum expression type promotor, tissue-specific promoter, tumor-specific promoters.The basic structure of promoter region: promotor is one section of DNA sequence dna being positioned at that structure gene 5 ' holds upstream, can activate RNA polymerase, make it combine exactly with template DNA and have the specificity of transcription initiation.Because the specific transcriptional of gene depends on that enzyme and promotor can form binary complex, therefore RNA polymerase how effectively to find promotor and to combine with it be the problem that first will solve in initiation of transcription.Have experiment to show, concerning many promotors, the speed that RNA polymerase combines with it is at least high than the random collision in pedesis 100 times.Transcriptional units (transcription unit) be one section from promotor to the DNA sequence dna that terminator (terminator) terminates, RNA polymerase is advanced along template from transcriptional start point, until terminator, transcribe out a RNA chain.In cell, a transcriptional units can be a gene, also can be several gene.Transcriptional start point refers to and the base on the corresponding DNA chain of nascent RNA chain first Nucleotide, and research confirmation is generally a purine.Often before starting point, namely the sequence of 5 ' end is called upstream (upstream), and the sequence of thereafter and namely 3 ' end is called downstream (downstream).When describing the position of base, generally use numeral, starting point is+1, and downstream direction is followed successively by+2 ,+3 ..., updrift side is followed successively by-1 ,-2 ,-3 ...The specificity of promotor is controlled by multiple element, and the controlling element played a crucial role at different sorts and different development stage is not identical, and the interaction between controlling element and trans-acting factor is also very complicated.Therefore foreign gene will be enable can efficiently to express specifically in animal tissues, and the selection of promotor is one of important condition building its expression vector.Research at present for transgenic animal field promoter activity adopts promoter deletion fragment activity analytical method usually.Namely according to the analysis of transcription factor forecasting software with determine promoter deletion clip size, PCR method is adopted to obtain the deletion fragment of different size.Then build promoter function analysis carrier by pGL3-Basic, detect promoter deletion fragment activity by Dual-Luciferase Activity Assay Kit.If occur that sp act changes between promoter deletion fragment, verify by base mutation.Zhao Dandan (2009) has carried out deletion mutantion research to ox α-actin promotor, prove that each deletion fragment of ox α-actin promotor all has very strong muscle specific, and prove wherein have one section of transcriptional activity relatively high, and moderate length (262bp).But its transcriptional activity is still lower compared with some strong promoters.How to search out a kind of can promote α-actin gene expression activity and the promotor with stronger muscle specific becomes to be applied to the transgenosis beef cattle production improving muscle yield a great problem being badly in need of solving? so, invent promotor and the application thereof of an ox α-actin gene, search out one and there is the active and skeletal muscle specificity of stronger startup and the promotor that can significantly improve α-actin activity of gene expression in ox bone arm muscle satellite cell is necessary.
Summary of the invention
In order to build a kind of can promote α-actin gene expression activity and there is the promotor of stronger muscle specific, to be applied to the transgenosis beef cattle production improving muscle yield, the invention provides promotor and the application thereof of an ox α-actin gene.The promotor of this ox α-actin gene and applications exploiting thereof have carried out promotor to ox α-actin promotor, and [fragmentation is studied, the same Zhao Dandan of method (2009), build the relatively high and ox α-actin promoter deletion fragment of moderate length (262bp) of one section of transcriptional activity, but the transcriptional activity of this fragment is still lower compared with some strong promoters.Proceed the research of PCR rite-directed mutagenesis, find the nucleotide sequence of the 170bp of one section, ox α-actin gene 262bp promoter fragment inside containing 3 positive controlling elements of SRE, different primers is used to carry out pcr amplification, obtain the nucleotide sequence of 2 170bp, the nucleotide sequence of 2 170bp is series at the upstream of 262bp promoter fragment, i.e. 170bp-170bp-262bp, is built into the promotor of a new α-actin gene.Finally, Dual-luciferase reportor systerm is adopted to verify the activity of this promotor in ox bone arm muscle satellite cell and bovine fibroblasts, find that its activity in the former even can more than CMV viral promotors, its activity in the latter is lower, thus the promotor demonstrating obtained α-actin gene has the active and skeletal muscle specificity of stronger startup.Meanwhile, this promotor can significantly improve the expression activity of α-actin gene in ox bone arm muscle satellite cell.Therefore, the new promotor built can be applied to as efficient specific skeletal muscle promotor and improve the transgenosis beef cattle production of muscle yield, thus be reached through build a kind of can promote α-actin gene expression activity and the promotor with stronger muscle specific to be applied to the object of the transgenosis beef cattle production improving muscle yield.
The technical solution adopted for the present invention to solve the technical problems is:
The nucleotide sequence of the promotor of an ox α-actin gene and the promoter fragment of application thereof is Seq ID No:l, concrete build and proof procedure as follows:
1. the clone of the inner 170bp of ox α-actin gene 262 promoter fragment 1. nucleic acid fragment: the positive recombinant plasmid pGL3-α-actin262 built with this laboratory is for template, the sequence of primer pair ox α-actin gene 262 promoter fragment transcription site upstream-224bp extremely containing multiple SRE controlling element between-55bp designed is utilized to carry out pcr amplification, obtain the nucleotide sequence of 170bp, and by this sequence designations be: α-actin170 1..Wherein α-actin170 1. in containing KpnI, BamHI double enzyme site.
2. the clone of the inner 170bp of ox α-actin gene 262 promoter fragment 2. nucleic acid fragment: the positive recombinant plasmid pGL3-α-actin262 built with this laboratory is for template, the sequence of primer pair ox α-actin gene 262 promoter fragment transcription site upstream-224bp extremely containing multiple SRE controlling element between-55bp designed is utilized to carry out pcr amplification, obtain the nucleotide sequence of 170bp, and by this sequence designations be: α-actin170 2..Wherein α-actin170 2. in containing BamHI, XbaI double enzyme site.
3. 1. build with α-actin170 pMD18-T cloning vector 2. containing ox α-actin170.(clone obtain carrier called after pMD18-T-Double-α-actin170-α-actin262): with BamHI, XbaI double digestion pMD18-T-α-actinp262 (this laboratory builds) and pMD18-T-α-actin170 2., reclaim the α-actin170 2. fragment of pMD18-T-α-actinp262 carrier large fragment and 170bp respectively, and the two is connected, build pMD18-T-α-actin170 2.-α-actinp262 recombinant plasmid, BamHI, XbaI double digestion identifies positive recombinant plasmid.With KpnI, BamHI double digestion pMD18-T-α-actin170,2.-α-actinp262 and pMD18-T-α-actin170 is 1., reclaim the α-actin170 1. fragment of pMD18-T-α-actin170 2.-α-actinp262 carrier large fragment and 170bp respectively, and the two is connected, build pMD18-T-Double-α-actin170-α-actin-262bp recombinant plasmid, KpnI, BamHI double digestion identifies positive recombinant plasmid.
4. containing ox α-actin170 1. with α-actin170 pGL3 expression vector establishment 2..(the carrier called after pGL3-Double-α-actin170-α-actin262 that clone obtains): with KpnI and HindIII double digestion pMD18-T-Double-α-actin170-α-actin-262bp and pGL3-Basic, reclaim Double-α-actin170 sequence and pGL3-Basic carrier large fragment that size is about 602bp, and the two is connected, build pGL3-Double-α-actin170-α-actin262 recombinant plasmid, KpnI and HindIII double digestion identifies positive recombinant plasmid.
5.pGL3-Double-α-actin170-α-actin262 expression vector carries out the transfection experiment of ox bone arm muscle satellite cell and bovine fibroblasts and the detection of Dual-luciferase reportor systerm, to measure promoter activity and the muscle specific thereof of improved Double-α-actin170-α-actin262 promoter fragment.Ox bone arm muscle satellite cell and bovine fibroblasts are cultivated.When the stand density of culturing cell arrives about 80%-90%, discard nutrient solution, rinse three times with the PBS without calcium ions and magnesium ions, add 0.25% tryptic digestion of 0.5mL, 37C places 1-2min.Under microscope during 80% cell rounding, add the Growth of Cells nutrient solution termination digestion of 5.5mL containing DMEM cell culture fluid+15% standard foetal calf serum, go down to posterity in 1: 2 ratio.Cell transfecting: transfected plasmids is with extracting without intracellular toxin plasmid extraction kit.Plasmid transfection reagent is polymine (PEI).Before transfection 24h by cell with 4-8 × 10
5the density paving of cells/well is with in 12 orifice plates.When complete adherent growth to 80% ~ 90% of cell merges, according to PEI transfection procedure step, different expression vector and sea pansy gene internal reference plasmid phRL-TK are distinguished cotransfection ox bone arm muscle satellite cell and bovine fibroblasts with the ratio of 50: 1 (mass ratioes), pGL3-Basic empty carrier and phRL-TK are using same condition as negative control group cotransfection cell, after transfection 4h, change differentiation culture liquid (2% horse serum+98%DMEM cell culture fluid) into, collecting cell after transfection 72h.Luciferase reporter gene detects, and is the expression level adopting luciferase reporter gene detection kit (Promega) to measure reporter gene.Experimental procedure is as follows: discard the cell culture fluid in 12 orifice plates, with PBS washed cell 2 times, adds 200ul cell pyrolysis liquid (1 × PLB), after room temperature places 15min, and collecting cell lysate.Get 20ul cell pyrolysis liquid in fluorometric assay pipe, add 100ul detection reagent (fireflyluciferase, LARII), mix rear Chemiluminescence Apparatus (FB 12Luminometer) and measure luminous value, value during record 10s, finally add the Stop & GLO reagent of 100ul, measure as interior target Renilla luciferase luminous value, detect value during 10s, both ratios are the relative reactivity RLA (Relative Luciferase Activity) of luciferase simultaneously.The numerical value of RLA is repeat experimental result mean+SD 3 times.Concrete operation step is see Promega detection kit specification sheets.
The promoter activity of 6.Double-α-actin170-α-actin262 promoter fragment and muscle specific thereof detect: adopt pGL3-basic, pGL3-α-actin262, pGL3-CMV in contrast, detects the promoter activity of Double-α-actin170-α-actin262 in pGL3-Double-α-actin170-α-actin262 expression vector.PGL3-basic, pGL3-α-actin262, these 4 kinds of expression vectors of pGL3-CMV and pGL3-Double-α-actin170-α-actin262 carry out the transfection of ox bone arm muscle satellite cell and bovine fibroblasts respectively, and 72 h before harvest cells also detect respective promoter activity and correct through internal reference renilla luciferase.It is active that detected result shows that pGL3-Double-α-actin170-α-actin262 has higher startup in ox bone arm muscle satellite cell, can be active more than the startup of pGL3-CMV, and the activity of pGL3-Double-α-actin170-α-actin262 in inoblast is lower, prove its good skeletal muscle specificity.
Beneficial effect of the present invention is: the present invention constructs the promoter fragment of a new α-actin gene, i.e. 170bp-170bp-262bp, the promoter fragment of the α-actin gene obtained has stronger startup activity and skeletal muscle specificity, simultaneously, this promoter fragment can significantly improve the expression activity of α-actin gene in ox bone arm muscle satellite cell, can be applied to the transgenosis beef cattle production improving muscle yield.
Accompanying drawing explanation
Below in conjunction with accompanying drawing, the present invention is further described.
Fig. 1 is the nucleic acid sequence of promoter (Seq ID No:1) of the promotor of an ox α-actin gene.
Fig. 2 is promoter activity and the muscle specific detected result figure thereof of the Double-α-actin170-α-actin-262bp promoter fragment of the promotor of an ox α-actin gene.1. bovine muscle satellite cell, 2. bovine fibroblasts, the 3. relative reactivity of luciferase in figure, 4.pGL3-basic, 5.pGL3-α-actin262,6.pGL3-CMV, 7.pGL3-Double-α-actin170-α-actin262.
Fig. 3 is the expression vector establishment schematic diagram of the promotor of an ox α-actin gene.
Embodiment
In the context of the present specification, unless specifically stated otherwise otherwise this specification sheets any term used has the implication that those skilled in the art understand in the art usually, and the experimental technique of unreceipted detailed conditions is conveniently test method or carry out according to the process specifications that supplier advises.
Embodiment one
1. the clone of the inner 170bp of ox α-actin gene 262 promoter fragment 1. nucleic acid fragment
The positive recombinant plasmid pGL3-α-actin262 built with this laboratory is for template, the primer (table 1) designed is utilized to carry out pcr amplification to the sequence containing multiple SRE controlling element between ox α-actin gene 262 promoter fragment transcription site upstream-224bp to-55bp, obtain the nucleotide sequence of 170bp, and by this sequence designations be: α-actin170 1..Wherein α-actin170 1. in containing KpnI, BamHI double enzyme site.PCR response procedures is: 94 DEG C of denaturation 5min, 94 DEG C of sex change 30s, annealing 30s, 72 DEG C of extensions, totally 35 circulations; 72 DEG C of ends extend 10min, are cooled to 4 DEG C of preservations.
Table 1 pcr amplification α-actin170 primer 1.
Be connected after α-actin170 1. PCR primer glue recovery purifying with pMD18-T cloning vector, adopt KpnI, BamHI double digestion to identify positive recombinant plasmid, deliver to the order-checking of Beijing Jin Weizhi genome company.The carrier called after pMD18-T-α-actin170 built 1..It is as follows that the glue of α-actin170 1. PCR primer reclaims the working method of purifying:
From sepharose, reclaim DNA fragmentation adopts Beijing TransGen company glue recovery test kit to carry out, and concrete grammar is as follows:
(1) Agarose plug containing object fragment band is cut off, put into the centrifuge tube of 1.5mL.
(2) add the sol solutions of 300 μ L by every 100mg agarose, be placed in 55 DEG C of water-bath 10min, agarose is dissolved completely, every 2min puts upside down mixing once.Loaded by agar liquid glucose immigration adsorption column after dissolving, room temperature leaves standstill 1-2min, and the centrifugal 30s of 12000rpm, removes waste liquid in collection tube.
(3) 700 μ L rinsing liquid rinsings, 10000rpm is centrifugal, and 30s outwells waste liquid, repeats once to outwell waste liquid again, centrifugal 30s under 10000rpm.
(4) be put into by adsorption column in new 1.5mL centrifuge tube, add elution buffer (ELution buffer) 30 μ L-50 μ L in adsorption column central authorities, room temperature places the centrifugal 1min of 1-2min, 10000rpm.DNA in 1.5mL centrifuge tube is stored in-20 DEG C.
The ligation system of table 2 PCR primer and pMD18-T cloning vector
The ligation system of PCR primer and pMD18-T expression vector is as shown in table 2, and (note: reaction system is 15 μ L, in this ligation system, the content that PCR reclaims product should be (3-10) with pMD18-T vector contg ratio: 1.About containing quality containing the PCR primer of the pMD18-T of 25ng, 7 μ L in 0.5uL carrier is 75-250ng, and 4 DEG C are spent the night).
2. the clone of the inner 170bp of ox α-actin gene 262 promoter fragment 2. nucleic acid fragment
The positive recombinant plasmid pGL3-α-actin262 built with this laboratory is for template, the primer (table 3) designed is utilized to carry out pcr amplification to the sequence containing multiple SRE controlling element between ox α-actin gene 262 promoter fragment transcription site upstream-224bp to-55bp, obtain the nucleotide sequence of 170bp, and by this sequence designations be: α-actin170 2..Wherein α-actin170 2. in containing BamHI, XbaI double enzyme site.PCR response procedures is: 94 DEG C of denaturation 5min, 94 DEG C of sex change 30s, annealing 30s, 72 DEG C of extensions, totally 35 circulations; 72 DEG C of ends extend 10min, are cooled to 4 DEG C of preservations.
Table 3 pcr amplification α-actin170 primer 2.
Be connected after α-actin170 2. PCR primer glue recovery purifying with pMD18-T cloning vector, adopt BamHI, XbaI double digestion to identify positive recombinant plasmid, deliver to the order-checking of Beijing Jin Weizhi genome company.The carrier called after pMD18-T-α-actin170 built 2..α-actin170 2. PCR primer glue reclaim purifying with pMD18-T-α-actin170 construction process is 1. identical above.
3. 1. build with α-acin170 pMD18-T cloning vector 2. containing ox α-actin170.(the carrier called after pMD 18-T-Double-α-actin170-α-actin262 that clone obtains)
With BamHI, XbaI double digestion pMD18-T-α-actinp262 (this laboratory builds) and pMD 18-T-α-actin170 2., reclaim the α-actin170 2. fragment of pMD 18-T-α-actinp262 carrier large fragment and 170bp respectively, and the two is connected, build pMD18-T-α-actin170 2.-α-actinp262 recombinant plasmid, BamHI, XbaI double digestion identifies positive recombinant plasmid.
With KpnI, BamHI double digestion pMD 18-T-α-actin170,2.-α-actinp262 and pMD 18-T-α-actin170 is 1., reclaim the α-actin170 1. fragment of pMD18-T-α-actin170 2.-α-actinp262 carrier large fragment and 170bp respectively, and the two is connected, build pMD18-T-Double-α-actin170-α-actin262 recombinant plasmid, KpnI, BamHI double digestion identifies positive recombinant plasmid.
4. containing ox α-actin170 1. with α-actin170 pGL3 expression vector establishment 2..(the carrier called after pGL3-Double-α-actin170-α-actin262 that clone obtains)
With KpnI and HindIII double digestion pMD18-T-Double-α-actin170-α-actin262 and pGL3-Basic, reclaim Double-α-actin170 sequence and pGL3-Basic carrier large fragment that size is about 602bp, and the two is connected, build pGL3-Double-α-actin170-α-actin262 recombinant plasmid, KpnI and HindIII double digestion identifies positive recombinant plasmid.5.pGL3-Double-α-actin170-α-actin262 expression vector carries out the transfection experiment of ox bone arm muscle satellite cell and bovine fibroblasts and the detection of Dual-luciferase reportor systerm, to measure promoter activity and the muscle specific thereof of improved Double-α-actin170-α-actin262 promoter fragment.
Ox bone arm muscle satellite cell and bovine fibroblasts are cultivated.When the stand density of culturing cell arrives about 80%-90%, discard nutrient solution, rinse three times with the PBS without calcium ions and magnesium ions, add 0.25% tryptic digestion of 0.5mL, place 1-2min for 37 DEG C.Under microscope during 80% cell rounding, add the Growth of Cells nutrient solution termination digestion of 5.5mL containing DMEM cell culture fluid+15% standard foetal calf serum, go down to posterity in 1: 2 ratio.
Cell transfecting: transfected plasmids is with extracting without intracellular toxin plasmid extraction kit.Plasmid transfection reagent is polymine (PEI).Before transfection 24h by cell with 4-8 × 10
5the density paving of cells/well is with in 12 orifice plates.When complete adherent growth to 80% ~ 90% of cell merges, according to PEI transfection procedure step, different expression vector and sea pansy gene internal reference plasmid phRL-TK are distinguished cotransfection ox bone arm muscle satellite cell and bovine fibroblasts with the ratio of 50: 1 (mass ratioes), pGL3-Basic empty carrier and phRL-TK are using same condition as negative control group cotransfection cell, after transfection 4h, change differentiation culture liquid (2% horse serum+98%DMEM cell culture fluid) into, collecting cell after transfection 72h.
Luciferase reporter gene detects, and is the expression level adopting luciferase reporter gene detection kit (Promega) to measure reporter gene.Experimental procedure is as follows: discard the cell culture fluid in 12 orifice plates, with PBS washed cell 2 times, adds 200ul cell pyrolysis liquid (1 × PLB), after room temperature places 15min, and collecting cell lysate.Get 20ul cell pyrolysis liquid in fluorometric assay pipe, add 100ul detection reagent (firefly luciferase, LARII), mix rear Chemiluminescence Apparatus (FB12Luminometer) and measure luminous value, value during record 10s, finally add the Stop & GLO reagent of 100ul, measure as interior target Renilla luciferase luminous value, detect value during 10s, both ratios are the relative reactivity RLA (Relative Luciferase Activity) of luciferase simultaneously.The numerical value of RLA is repeat experimental result mean+SD 3 times.Concrete operation step is see Promega detection kit specification sheets.
The preparation of 1 × PLB: according to required Dosage calculation, is added to 5 × PLB of 1 times of volume in the distilled water of 4 times of volumes, mixes, 4 DEG C of preservations.This solution is prepared fresh before every use.
LAR II: by fluoroscopic examination damping fluid (Luciferase Assay Buffer II) dissolved freeze-dried powder, can one month be preserved at-20 DEG C after mixing, 1 year can be preserved at-70 DEG C.
Stop &
reagent: get appropriate 50 × Stop &
substrate joins the Stop & of requirement
in Buffer, final concentration is made to become 1 times of concentration.
Sample preparation: discard the cell culture fluid in 12 orifice plates, adds 1 × PBS and cleans culturing cell, wash 2 times.Remove scavenging solution, add 200 μ l Fresh lysate (1 × PLB).Room temperature places 15min, and collecting cell lysate is in centrifuge tube.The centrifugal 5min of 12000r/min, get 20 μ L supernatant liquors in clean centrifuge tube, sample retention is in-20 DEG C.
The promoter activity of 6.Double-α-actin170-α-actin-62 promoter fragment and muscle specific thereof detect
Adopt pGL3-basic, pGL3-α-actin262, pGL3-CMV in contrast, detects the promoter activity of Double-α-actin170-α-actin262 in pGL3-Double-α-actin170-α-actin262 expression vector.PGL3-basic, pGL3-α-actin262, these 4 kinds of expression vectors of pGL3-CMV and pGL3-Double-α-actin170-α-actin262 carry out the transfection of ox bone arm muscle satellite cell and bovine fibroblasts respectively, and 72 h before harvest cells also detect respective promoter activity and correct through internal reference renilla luciferase.It is active that detected result shows that pGL3-Double-α-actin170-α-actin262 has higher startup in ox bone arm muscle satellite cell, can be active more than the startup of pGL3-CMV, and the activity of pGL3-Double-α-actin170-α-actin262 in inoblast is lower, prove its good skeletal muscle specificity.
More than show and describe ultimate principle of the present invention and principal character and advantage of the present invention.The technician of the industry should be appreciated that; the present invention is not restricted to the described embodiments; what describe in above-described embodiment and specification sheets just illustrates principle of the present invention; without departing from the spirit and scope of the present invention; the present invention also has various changes and modifications; these changes and improvements all fall in the claimed scope of the invention, and application claims protection domain is defined by its equivalent of appending claims.
Claims (1)
1. the promotor of an ox α-actin gene and application thereof, it is characterized in that the nucleotide sequence of the 170bp of one section, ox α-actin gene 262bp promoter fragment inside containing 3 positive controlling elements of SRE, different primers is used to carry out pcr amplification, obtain the nucleotide sequence of 2 170bp, the nucleotide sequence of 2 170bp is series at the upstream of 262bp promoter fragment, i.e. 170bp-170bp-262bp, be built into the promoter fragment of a α-actin gene, its nucleotide sequence is as shown in Seq ID No:1.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1432577A (en) * | 2002-01-14 | 2003-07-30 | 李宁 | Sheep growth hormone releasing hormone gene and its expression product and application |
| CN102127546A (en) * | 2010-11-24 | 2011-07-20 | 山东农业大学 | Skeletal muscle specificity actin promoter and applications thereof |
| CN103074370A (en) * | 2011-10-26 | 2013-05-01 | 南京医科大学 | Transgenic vector used for improving pig lean meat percentage by specifically expressing Follistatin in muscle tissues |
| CN103194450A (en) * | 2013-04-02 | 2013-07-10 | 东北农业大学 | Method for increasing activity of myogenin (MyoG) gene promoter |
-
2015
- 2015-01-29 CN CN201510063159.2A patent/CN104651365A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1432577A (en) * | 2002-01-14 | 2003-07-30 | 李宁 | Sheep growth hormone releasing hormone gene and its expression product and application |
| CN102127546A (en) * | 2010-11-24 | 2011-07-20 | 山东农业大学 | Skeletal muscle specificity actin promoter and applications thereof |
| CN103074370A (en) * | 2011-10-26 | 2013-05-01 | 南京医科大学 | Transgenic vector used for improving pig lean meat percentage by specifically expressing Follistatin in muscle tissues |
| CN103194450A (en) * | 2013-04-02 | 2013-07-10 | 东北农业大学 | Method for increasing activity of myogenin (MyoG) gene promoter |
Non-Patent Citations (1)
| Title |
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| 孟庆勇: "肌肉注射GHRH表达质粒促进动物生长在大鼠、猪和绵羊中的研究", 《中国农业大学博士论文》 * |
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