CN102127546A - Skeletal muscle specificity actin promoter and applications thereof - Google Patents

Abstract

The invention belongs to the biotechnology field and relates to a skeletal muscle specificity promoter and the applications thereof, in particular to a skeletal muscle specificity expression gene 5' control region specificity promoter and the applications thereof to transgenic pigs. The promoter has promotion specificity only in skeletal muscle and has no promotion activity in various cells except for skeletal muscle so that the gene promoter only specifically promotes the expression of a downstream gene in the skeletal muscle. Accordingly, the invention can meet the need for the specific expression of a target gene in the skeletal muscle and provides an important element for improving pork quality through a transgene technology.

Description

A kind of skeletal muscle specificity actin promotor and application thereof

Technical field

The present invention relates to the molecular genetics field, be specifically related to a kind of skeletal muscle different expression gene 5 ' control region specificity promoter and the application in transgenic pig thereof.

Background technology

Time and level that eukaryotic gene is expressed are carried out in proper order in strict accordance with growth.The adjusting that some specific gene is expressed is incorporated into the control region related elements by the transcription factor specificity, becomes the committed step of transcription initiation, and finally causes the time and the space expression of gene.The series family gene that coding is organized differential protein is in strict accordance with tissue specificity and the expression of etap property, for gene expression regulation mechanism provides the excellent research pattern.In the transgenic animal of specific high-efficiency expression, the specificity promoter that obtains the energy widespread use is extremely important.But the specificity of promotor is controlled by a plurality of elements, and the controlling element that plays a crucial role at different sorts and different development stage is also inequality, and the interaction between controlling element and the trans-acting factor is also very complicated.Foreign gene can be expressed on differential high efficient ground in animal tissues, must structure one can specific high-efficiency expression the animal expression carrier, wherein the selection of promotor is one of important condition.

Actin muscle (actin) is the main component of myofilament.The Actin muscle that has been found that in Mammals has 6 kinds of different subtypes, can be divided into alpha according to the iso-electric point difference, beta, gamma three classes, wherein alpha skeletal muscle type Actin muscle (alpha actin) belongs to contractile protein, it is the easypro active basic substance that contracts of Skeletal Muscle Cell, also be the distinctive albumen of skeletal muscle, account for 12% of skeletal muscle total protein, pig alpha actin gene is positioned on No. 14 karyomit(e), form by 7 exons and 6 introns, 377 amino acid of encoding, by cloning this gene promoter area and in its starting characteristic of analyzed in vitro, utilizing correlated series to make up the skeletal muscle specific expression vector is a kind of effective means, but above-mentioned research all also is in the starting stage, does not all play promoter action preferably for the cultivation and the quality raising of transgenic pig.

Summary of the invention

Based on above-mentioned reason, the present inventor is through discovering that pig alpha actin gene 5 ' control region has skeletal muscle specificity starting characteristic, and a kind of skeletal muscle specific expressing promoter carrier that utilized this characteristics design, this promotor can be used in the cultivation of transgenic pig.This promoter vector has skeletal muscle specificity starting characteristic, and it is active all not have startup in the various kinds of cell except that skeletal muscle, as seen this promotor starts the expression of downstream gene specifically in skeletal muscle, can satisfy goal gene specific expressed requirement in skeletal muscle, for transgene carrier provides a kind of important element.

Skeletal muscle different expression gene 5 ' control region specificity promoter provided by the present invention, its gene order is shown in Seq ID No:1;

Except above-mentioned promotor, can also be this promoter variants or fragment, all have the gene order shown in Seq ID No:1.

" variant " is meant pig alpha actin promotor Seq ID No:1 sequence carried out any replacement, variation, modification, replacement, the disappearance of one or more base or the sequence that interpolation produced, and this sequence still shows the activity that is similar to Seq ID No:1 dna sequence dna.

" fragment " is meant one or more zone of basic pig alpha actin promotor Seq ID No:1 sequence, and it still is similar to the activity of basic dna sequence dna.

Promotor or variant with above-mentioned sequence, perhaps fragment, has skeletal muscle specificity starting characteristic, and it is active all not have startup in the various kinds of cell outside skeletal muscle, as seen this gene promoter starts the expression of downstream gene specifically in skeletal muscle, can satisfy goal gene specific expressed requirement in skeletal muscle, and can be applied in the cultivation of transgenic pig.

When using above-mentioned promotor, the general recombinant nucleic acid sequence that adopts contains above-mentioned promotor or its variant, perhaps fragment in this sequence, so just can make in the goal gene in promotor downstream specific expressed in skeletal muscle, as analyzing, exploitation, transforming skeletal muscle The Characteristic Study model.Can also utilize existing molecular biology operative technique to obtain to comprise the recombinant nucleic acid of goal gene, obtain transgenic embryos, be used to set up transgenic animal by gene leading-in techniques such as microinjections.Goal gene in the recombinant nucleic acid sequence, contain following at least a functional gene, mainly comprise and improve the meat quality gene, accelerate speed of growth gene, disease-resistant gene, improve the gene of the price of deed and the gene of production certain drug, so just can obtain outside the skeletal muscle specificity starting characteristic, improve the performance of this recombinant nucleic acid sequence, for the cultivation of transgenic pig provides the more function direction, increase its additional output value,, improve the meat nutritive value as desaturation fatty acid enzyme gene is expressed the content that can specificity improves unsaturated fatty acids in the skeletal muscle under this promotor instructs.

After having obtained above-mentioned recombinant nucleic acid sequence, can also set up special expression system with it, so that better application is in the cultivating process of transgenic pig.Goal gene is recombinated to this promotor downstream, changing technology over to by gene makes control region and goal gene be integrated in the karyomit(e) jointly, obtain transgenic animal, goal gene is only expressed in skeletal muscle under this promotor effect, in other histoorgans, do not express, reduce the influence of goal gene, improve genetically modified effect animal.

The present invention mainly determines the startup feature of this promotor by the reporter gene analysis.At first make up no promotor green fluorescent protein carrier,, insert internal ribosome entry site (IRES) in the green fluorescence protein gene upstream for guaranteeing the reporter gene correct translation.Obtain specific expressed alpha Actin muscle 5 ' control region in the skeletal muscle by PCR or fragment technology such as catch then, length is 3.7kb, insert upstream, internal ribosome site, obtain the promotor reporter gene and analyze carrier, CMV promotor (589bp) is connected in the empty carrier as positive control the negative contrast of empty carrier.After plasmid removes intracellular toxin, transfection C2C12 mouse muscle-forming cell system, former generation myocardial cell of transfection pig and pig renal epithelial cell are that PK15 is contrast simultaneously, change behind the transfection 24h and contain the inducing culture of horse serum to sarcoplast, the inducing cell differentiation is merged, observe the egfp expression situation with inverted fluorescence microscope behind the 48h, analyze the promotor characteristic.

When above-mentioned promotor, after perhaps recombinant nucleic acid sequence or expression system are applied in the cultivation of transgenic pig, the effect of cultivation can effectively be provided, and interpolation by various functioning genes, make transgenic pig can obtain better meat quality, the higher speed of growth, better disease resistance and reduction feeding cost, can also in the production of certain drug, obtain using more widely, good reference value be arranged for the research in this field.

Description of drawings

Fig. 1 is ActinF and ActinR primer PCR amplification alpha actin promoter fragment electrophorogram, and swimming lane 1,2 is the PCR product;

Fig. 2 is no promotor CMV Enhancer IRES AcGFP carrier collection of illustrative plates;

Fig. 3 is a transfection CMV Enhancer alpha actin promoter pIRES-AcGFP 48h mouse C2C12 cell photo,

The left side is a photo under 80 times of light microscopics, and cell state is good, and the right side is blue-light excited photo down, and visible GFP expresses;

Fig. 4 is a transfection CMV Enhancer alpha actin promoter pIRES-AcGFP 48h Pigs Hearts cell photo,

The left side is a photo under 40 times of light microscopics, and cell state is good, and the right side is blue-light excited photo down, and no GFP expresses;

Fig. 5 is a transfection CMV Promoter pIRES-AcGFP 48h Pigs Hearts cell photo,

The left side is a photo under 40 times of light microscopics, and cell state is good, and the right side is blue-light excited photo down, and visible GFP expresses;

Fig. 6 is a transfection CMV Enhancer alpha actin promoter pIRES-AcGFP 48h porcine kidney cell photo,

The left side is a photo under 40 times of light microscopics, and cell state is good, and the right side is blue-light excited photo down, and no GFP expresses;

Fig. 7 is a transfection CMV Promoter pIRES-AcGFP 48h porcine kidney cell photo,

The left side is a photo under 40 times of light microscopics, and cell state is good, and the right side is blue-light excited photo down, and visible GFP expresses;

Embodiment

In the context of the present specification, unless specialize otherwise the used any term of this specification sheets has those skilled in the art's implication of common sense in the art, and the experimental technique of unreceipted detailed conditions is to carry out according to the routine test method or according to the process specifications that supplier advised.

Embodiment 1 no promotor IRES-AcGFP vector construction

ATG is to the influence of reporter gene translation in the analytical sequence, guarantee the correct expression of downstream reporter gene GFP, the no promotor IRES-AcGFP carrier of special structure, this carrier comprises an internal ribosome and enters element, this method helps keeping the controlling element in transcription initiation site downstream, can obtain bigger control region analystal section.Concrete grammar is cut with Ase I, Nhe I enzyme for being skeleton with pIRES-AcGFP, reclaims the 4723bp fragment, obtains not having the fragment of CMV promotor; Pcr amplification CMVEnhancer element, downstream primer adds XbaI enzyme cutting site and protectiveness base, and overstriking is represented.CMV Enhancer Forward gene order is shown in Seq ID No:2, and CMV Enhancer Reverse gene order is shown in Seq ID No:3.

According to following system preparation PCR reaction solution

2×PrimeSTAR ^TM?GC?Buffer 10μl

dNTP?Mixture(2.5mM?each) 2μl

CMV?Enhancer?Forward 0.3μM(final?conc.)

CMV?Enhancer?Reverse 0.3μM(final?conc.)

PIRES-AcGFP plasmid DNA 100ng

PrimeSTAR ^TM?HS?DNA?Polymerase(2.5U/μl) 0.2μl

H ₂O complements to 20 μ l

The PCR program setting is as follows

Product after the amplification is mixed sample to the 1%TAE sepharose with 6 * Loading buffer, cut glue behind the 5V/cm electrophoresis 20min and reclaim the purpose fragment, obtain purified product by the operation of Axygen sepharose purification kit purifying specification sheets, cut purified product with Ase I (Fermentas) and Xba I (Fermentas) enzyme, agarose was cut glue and is reclaimed the CMV Enhancer that obtains sticky end after enzyme was cut, CMV Enhancer element is connected with Rapid DNA Ligation (Fermentas) with carrier after enzyme is cut, the clone transforms, and obtains to contain the promoterless pIRES-AcGFP of CMV Ehancer.Complete CMV promotor forward is connected in this carrier, obtains the positive control carrier of this carrier.The bacterium liquid that will comprise negative control and positive control carrier is seeded to respectively according to 1: 500 ratio and contains in the kantlex 50 μ g/ml LB substratum, and 200rpm acutely rocks 14h.4 ℃ of 6000 * g collect thalline to the 50ml centrifuge tube, are used to not have the intracellular toxin plasmid and extract, and concrete operations are carried out according to EndoFree Plasmid Maxi (QIAGEN) specification sheets, and the plasmid A260/280 that purifying obtains is between 1.8-1.9.ActinF and ActinR primer PCR amplification alpha actin promoter fragment electrophorogram such as Fig. 1.

The structure of embodiment 2 promotor reporter gene carriers

Genome extracts: get the LargeYorkshire ear tissue, carry out extracting genome DNA according to TIANamp genomic DNA (day root) test kit specification sheets.

Follow according to NCBI (Gene ID:100154254) and design following primer, the ActinF gene order is shown in Seq ID No:4, and the ActinR gene order is shown in Seq ID No:5, and it has comprised the scope from transcription initiation site downstream 832bp to upstream 2885bp.

According to following system preparation PCR reaction solution

2×PrimeSTAR ^TM?GC?Buffer 10μl

dNTP?Mixture(2.5mM?each) 2μl

ActinF 0.3μM(final?conc.)

ActinR 0.3μM(final?conc.)

Pig genomic dna 100ng

PrimeSTAR ^TM?HS?DNA?Polymerase(2.5U/μl) 0.2μl

H ₂O complements to 20 μ l

The PCR program setting is as follows

Product after the amplification is mixed sample to the 0.5%TAE sepharose with 6 * Loading buffer, cut glue behind the 5V/cm electrophoresis 20min and reclaim big fragment, obtain purified product by the operation of Axygen sepharose purification kit purifying specification sheets, purified product is connected in the pJET1.2 cloning vector (Fermentas), the clone transforms DH5 α, the order-checking of picking list bacterium colony, errorless back acquisition alpha actin promotor-pJET1.2 plasmid checks order, cut this plasmid with Bgl II (Fermentas) enzyme, agarose is cut glue and is reclaimed the alphaactin promotor shown in Seq ID No:1.

Promoterless pIRES-AcGFP carrier is cut with Bgl II (Fermentas) enzyme, adds FastAP simultaneously ^TMThermosensitive Alkaline Phosphatase (Fermentas) carries out dephosphorylation to be handled, alpha actin promotor is connected with dephosphorylized carrier with Rapid DNA Ligation (Fermentas), and conversion DH5 α, choose the forward and reverse of bacterium checking insertion behind the coated plate, the bacterium liquid that will comprise correct direction of insertion is seeded to according to 1: 500 ratio and contains in the kantlex 50 μ g/ml LB liquid nutrient mediums, and 200rpm acutely rocks 14h.4 ℃ of 6000 * g collect thalline to the 50ml centrifuge tube, are used to not have the intracellular toxin plasmid and extract, and concrete operations are carried out according to EndoFree Plasmid Maxi (QIAGEN) specification sheets, and the plasmid A260/280 that purifying obtains is between 1.8-1.9.Obtain recombinant nucleic acid CMV Enhancer alpha actin Promoter pIRES-AcGFP carrier, and utilize the method for transient transfection cell on cell levels, to verify the start-up characteristic of alpha actin promotor.

Embodiment 3 cell cultures and transfection

The two age in days two-way cross piggys that will be born are put to death, aseptic condition takes out heart, kidney down to the physiological saline of room temperature, heart is transferred among the 1 * PBS (Hyclone) of 37 ℃ of preheatings after the rinsing in physiological saline gently, blood stains are removed in flushing, tear pericardium off, cut off the atrium, ventricle is shredded into 1mm ³Fritter, add the resuspended tissue block of 1 * PBS (Hyclone), leave standstill 3min, remove supernatant, add 4 ℃ of 0.05% pancreatin (Hyclone), place 4 ℃ of refrigerators to hatch, turn upside down behind the 3h 1 time, remove pancreatin behind the 6h, place 37 ℃ to hatch 20min tissue block, add 37 ℃ of 10ml perfect medium DMEM/F12 and (contain 20%FBS, penicillin 100U/ml, Streptomycin sulphate 100 μ g/ml), blow and beat tissue block gently with suction pipe, disappear, leave standstill 30sec until tissue block, get the upper strata cell suspension and measure cell viability and counting, cell is pressed 1 * 10 ⁴Individual/ml is seeded in the perfect medium, places 37 ℃, 5%CO ₂Cultivate.

With the kidney of aseptic taking-up, in physiological saline, be transferred among 1 * PBS of 37 ℃ of preheatings after the rinsing gently, blood stains are removed in flushing, and fat tears surperficial manadesma off, cuts off kidney, cuts off renal plevis and only stays the about 1g of renal cortex, and it is shredded into 1mm ³Fritter, add the resuspended tissue block of 40ml 1 * PBS (Hyclone), leave standstill 3min, remove supernatant, add 37 ℃ of 0.1% collagenase of 30ml, turn upside down 10 times, place 37 ℃ to hatch 60min, add 2ml foetal calf serum (Gibco), blow and beat tissue block gently with suction pipe, disappear, leave standstill 1min until tissue block, get the upper strata cell suspension and measure cell viability and counting, cell is pressed 1 * 10 ⁴Individual/ml is seeded in the perfect medium, places 37 ℃, 5%CO ₂Cultivate.

Mouse muscle-forming cell is that C2C12 cultivates in the DMEM that contains 10% foetal calf serum (Gibco) (Hyclone) substratum, and culture condition is 37 ℃, 5%CO ₂, keep cell degree of converging 50%, inducing culture is for containing the DMEM substratum of 2% horse serum (Hyclone).

To treat cells transfected with 1 * PBS (Hyclone) rinsing attached cell twice, and add 0.05% pancreatin (Hyclone) of 37 ℃ of preheatings, digestion 7min adds the perfect medium re-suspended cell of antibiotic-free, and every hole is by 1 * 10 after the cell counting ⁴Be seeded to 24 orifice plates, with 37 ℃ of serum-free antibiotic-free DMEM (Hyclone) substratum washed cell once, every hole adds the perfect medium of the fresh antibiotic-free of 500 μ l, carries out transfection behind the 12h behind the 24h, and this moment, cell degree of converging was 90%～95%.

With positive control support C MV Promoter pIRES-AcGFP, the promoterless CMV Enhancer of negative control carrier pIRES-AcGFP, experiment support C MV Enhancer alpha actin three kinds of plasmids of Promoter pIRES-AcGFP transfection simultaneously mouse muscle-forming cell is C2C12, the pig myocardium cell, porcine kidney cell, every hole is diluted to the 750ng plasmid among the 100 μ l opti-MEM, adds 0.75 μ l Lipofectamine ^TMPLUS (Invitrogen) is mixing thoroughly, adds 4 μ l Lipofectamine behind the 5min ^TMLTX (Invitrogen) transfection reagent is put upside down mixing, hatches for 25 ℃, adds in each hole mixing behind the 30min gently.Change inducing culture behind the transfection 12h to C2C12 clone, the inducing cell differentiation is merged.Observe down the GFP expression with the fluorescence inverted microscope behind the transfection 48h: the transfection efficiency of positive control carrier is at 30% (Fig. 5, Fig. 7), redgreen fluorescent protein expression after the transfection of negative control carrier.Behind the transfection CMV Enhancer alpha actin Promoter pIRES-AcGFP carrier, having only through inducing the mouse muscle-forming cell of differentiation is that C2C12 has egfp expression (Fig. 3), equal redgreen fluorescent protein expression (Fig. 4, Fig. 6) in cardiac muscle and nephrocyte.

Transfection results only show in sophisticated myocyte pig alpha actin promotor just have start active, in another kind of voluntary muscle myocardial cell, do not start active, in renal epithelial cell, there is not the activity of startup yet, show that pig alpha actin promotor has the skeletal muscle specificity and starts active, can the driving purposes gene specific expressed at skeletal muscle.

Embodiment 4alpha actin promoter variants function

According to the explanation of manufacturers, utilize Stratagene QuikChange ^TMRite-directed mutagenesis test kit (Stratagene), the design mutant primer makes up this promoter variants, comprises the disappearance of nucleic acid, replaces and insertion.The promotor of change is by sequencing checking sudden change.Promotor after the sudden change is recombinated to the goal gene upstream driving purposes expression of gene.

The application of embodiment 5 series connection alpha actin promotors

Follow according to NCBI (Gene ID:100154254) and design following primer, the upstream and downstream primer is introduced Xho I, EcoR I restriction enzyme site and protection base respectively.Alpha actin2F gene order is shown in Seq ID No:6, and alpha actin2R gene order is shown in Seq IDNo:7.

The PCR program setting is as follows

Product after the amplification is mixed sample to the 0.5%TAE sepharose with 6 * Loading buffer, cut glue behind the 5V/cm electrophoresis 20min and reclaim the 3.7kb fragment, obtain purified product by the operation of Axygen sepharose purification kit purifying specification sheets.Cut the PCR purified product and reclaim enzyme and cut product with XhoI (Fermentas) enzyme.

Alpha actin Promoter pIRES-AcGFP carrier is cut with Xho I (Fermentas) enzyme, added FastAP simultaneously ^TMThermosensitiveAlkaline Phosphatase (Fermentas) carries out dephosphorylation to be handled, Xho I enzyme is cut the ALPHAACTIN control region that purifying crosses to be connected with dephosphorylized carrier with Rapid DNA Ligation (Fermentas), and conversion DH5 α, choose the forward and reverse of bacterium checking insertion behind the coated plate, the bacterium liquid that will comprise correct direction of insertion is seeded to according to 1: 500 ratio and contains in the kantlex 50 μ l g/ml LB liquid nutrient mediums, and 200rpm acutely rocks 14h.4 ℃ of 6000 * g collect thalline to the 50ml centrifuge tube, are used to not have the intracellular toxin plasmid and extract, and concrete operations are carried out according to EndoFree Plasmid Maxi (OIAGEN) specification sheets, and the plasmid A260/280 that purifying obtains is between 1.8-1.9.Obtain recombinant nucleic acid CMV Enhancer double alpha actin Promoter pIRES-AcGFP carrier, and utilize the method for transient transfection cell on cell levels, to verify the start-up characteristic of two alpha actin promotors.

Embodiment 6 alpha actin promoter fragments are used

Follow according to NCBI (Gene ID:100154254) and design following primer, alpha actin3F gene order is shown in Seq ID No:8, and alpha actin3R gene order is shown in Seq ID No:9.

According to following system preparation PCR reaction solution

2×PrimeSTAR ^TM?GC?Buffer 10μl

dNTP?Mixture(2.5mM?each) 2μl

alpha?actin2F 0.3μM(final?conc.)

alpha?actin2R 0.3μM(final?conc.)

Pig genomic dna 100ng

PrimeSTAR ^TM?HS?DNA?Polymerase(2.5U/μl) 0.2μl

H ₂O complements to 20 μ l

The PCR program setting is as follows

Product after the amplification is mixed sample to the 0.5%TAE sepharose with 6 * Loading buffer, cut glue behind the 5V/cm electrophoresis 20min and reclaim the 467bp fragment, gene order is shown in Seq ID No:10, obtain purified product by the operation of Axygen sepharose purification kit purifying specification sheets, purified product is connected in the pJET1.2 cloning vector (Fermentas), the clone transforms DH5 α, the order-checking of picking list bacterium colony, errorless back acquisition alpha actin promoter fragment-pJET1.2 plasmid checks order, cut this plasmid with Bgl II (Fermentas) enzyme, agarose is cut glue and is reclaimed 500bp place fragment.

Promoterless pIRES-AcGFP carrier is cut with Bgl II (Fermentas) enzyme, adds FastAP simultaneously ^TMThermosensitive Alkaline Phosphatase (Fermentas) carries out dephosphorylation to be handled, alpha actin 5 ' control region fragment is connected with dephosphorylized carrier with Rapid DNA Ligation (Fermentas), and conversion DH5 α, choose the forward and reverse of bacterium checking insertion behind the coated plate, the bacterium liquid that will comprise correct direction of insertion is seeded to according to 1: 500 ratio and contains in the kantlex 50 μ g/ml LB liquid nutrient mediums, and 200rpm acutely rocks 14h.4 ℃ of 6000 * g collect thalline to the 50ml centrifuge tube, are used to not have the intracellular toxin plasmid and extract, and concrete operations are carried out according to EndoFree Plasmid Maxi (OIAGEN) specification sheets, and the plasmid A260/280 that purifying obtains is between 1.8-1.9.Obtain recombinant nucleic acid CMV Enhancer alpha actin Promoter pIRES-AcGFP carrier, and utilize the method for transient transfection cell on cell levels, to verify the start-up characteristic of alpha actin promoter fragment.

＜110〉Shandong Agricultural University

＜120〉a kind of skeletal muscle specificity promoter and application thereof

<160>10

<210>1

<211>3717

<212>DNA

＜213〉artificial sequence

<400>1

gggatgatag?ataatagttg?ccagtgaagc?catgactgca?tttaagatgt?atttgtccct 60

tcagcagaaa?agatcccttg?aagatctgtt?atgttccaga?atacagaagg?tgtgggagaa 120

ggttaaaggg?agataggaag?gtcgcccaag?cccctgcacg?accccagctg?ctttgggacc 180

tgagacgagc?tctctggctg?gttgggcctg?gctttgctta?tttttcaagc?tgcctcgggt 240

atcacctgtc?ttgtgtacct?caggatagtt?aattaatggg?aacaacgcga?aagcgtatga 300

agcattttat?gaattcacat?ctaagtaggt?agaaggctca?tactgtgcaa?actcggggtc 360

tgcagatatg?tgaggagctg?aggggacatg?gctgaaagag?gggtgtgggc?acgctggctg 420

ggtcatggca?gagggcctgt?caggatcaag?cgctgaggct?gcacctgaaa?acttagaagc 480

aggagctctg?tcaggcatac?cctgcatggc?tgacctttcc?tcaccacagt?tctcaaaagg 540

ctttgcgagc?agtaacccgc?ttactcaggc?gagtgcaagc?tgctgctgtg?tccccaaagt 600

gtacatgtat?ggaggcggcc?ttccacctgc?ccctcaggtc?tgtccctaag?ccaatgagaa 660

ttcagcagca?ccagaatggc?ctggagctgc?cccatggtga?cagtggggga?gtatttaaag 720

tggcccaacc?agggctgaga?gcagacatct?ccactgggca?tctcagggcc?catctcccca 780

aggtgtgctg?gactagctag?atctctgcta?gcctgtgagt?gttgtgttca?tagcacttca 840

tgtgtgcttg?aagccaatta?gaaaagcttg?ctataggaga?gaaaagagct?gcactgagca 900

ccctccttcc?cctttaaatg?tcaacagatt?aggagtcagt?gaatgacagc?acacctcttg 960

ctaccttaga?gaccaaaatt?taagctactc?cccttaagct?atagctagag?tgcacctgcc 1020

agtgtcttta?gtccccactg?atggaacagg?acccaaggta?ttgaagatgg?aacatagtta 1080

ttcattcatc?ctctaattta?aaaagctgga?tatgctgtac?agcagaaatt?gacggaacaa 1140

tgtaaatcaa?ctataacaga?agaaataaaa?acctgggggg?aaagaagctg?actatgaaac 1200

cccaggagct?ttctacatgg?gcctggactc?accaaactct?ttattttgta?atggacttct 1260

gacattttta?ggaagggctg?tcctgatgtg?ggctatagaa?gagggtttca?catgcttctt 1320

caagaggacc?cacactgtcc?cagttgctga?gtcccaccac?cagatgctag?tggcagctat 1380

ttggggaaca?cttaggcact?acaaaaaaat?gagtgattcc?attctggctc?acaccatatc 1440

cctgatgtac?cccttaaagc?atgtcactga?gttcatcaca?gaaaattgtt?tcccctgtgc 1500

cttccacaac?aaggttagag?ctgtccttgg?ggccagggga?agggggcagg?gagtgagaag 1560

caccaactgg?ataacctcct?ctgaccccca?ctccacctta?ccataagtag?atccaaatcc 1620

ttctagaaaa?ttaggaaggc?atatccccat?atatcagcga?tataaataga?actgcttcag 1680

cgctctggta?gacggtgact?ctccaaggtg?gactgggagg?cagcctggcc?ttggctgggc 1740

atcgtcctct?aaatagaaag?atgaacttgt?tcagcctttc?cagaaggaaa?actgctgccc 1800

agcctacagt?gcaacgtcct?tgtcttccat?ctggaggaag?cacgggtgac?atatcatcta 1860

gtaagggcac?ctctctgttt?ccacctccag?gtcgaggggt?gtgaccctta?cttctcagcc 1920

tcaagggagg?gacactcaac?cccccaaaaa?gacatgaggg?cgctcagctc?ggcccaccgc 1980

accccggacc?ggagccgtca?ccccccgaaa?ttcactccct?tcacaagccc?ccaagcgcgt 2040

tctctggtgc?ggactgctcc?ggggccctgg?ctttgtgccc?agcgttgtca?gagccaccgc 2100

cctgagcctg?tccccgggag?ccccgcgcct?cctcccaccg?ctccgctctc?gcgccccgcg 2160

gccagttgtc?tgccccgaga?cagctgcgcg?ccctcccgct?gccggtggcc?ctctccggtg 2220

ggggtgggga?ccgacagggt?cagccctccg?gatccggggc?gctccgggta?gcggggagaa 2280

gtgatcgctg?gggagctggg?ggaggggtcg?ccttcctgcc?ctacccagga?ctccgggtgc 2340

gaccgctcct?ctatctctcc?agcccaccac?cactccacca?cttggacacg?tctccctcct 2400

ccctggagtc?gctctagagg?gtttgggggt?ctgagtaaag?aacccgaagt?agggatacag 2460

tgtggcggca?ccttccagag?gccccgggcg?cagggtagac?cggggcgggg?cggcccgcgg 2520

acaggtgcag?ccccaggcgc?aggcgcactc?gcgcctcccg?gcgcaggcgg?tgaacctcgc 2580

cccaccccag?cccctccggg?gggcagctgg?gccgggtcgg?gaggggccca?ccagcccggg 2640

agacactcca?tatacggcca?ggcccgcttt?acctgggctc?cggccaggcc?gctccttctt 2700

tggtcagcac?aggggacccg?ggcgggggcc?caggccgcta?acccgccggg?ggagggggct 2760

ccagtgccca?acacccaaat?atggctcgag?aaggggagcg?acattccagt?gaggcggctc 2820

ggggggagaa?cccgcgggct?atataaaacc?tgagcgtggg?gaccagcggc?caccgcagcg 2880

gacagcgccg?agagaagcct?cgcttccctc?ccgcggcgac?cagggcccca?gccggagagc 2940

agcaggtgta?gccacccgcc?caggtagaca?agagtgggat?ggggacaggg?agacagggca 3000

ctgtcgaggg?gagcccagag?agcttccggg?agcccagccg?accccatcta?ttagccgcga 3060

ccctgcctcc?tcggtgctgc?tcggtcccca?gagccacatg?cgctttaaag?aggagacagg 3120

aagcagtcga?cctttggaaa?cgtctccaaa?tttattccct?gcccctttgg?atttctctac 3180

ctacacttag?tatgtgtcct?ggtagatgaa?cgtttgactt?tagtagtccc?cagggccaac 3240

cgagccaaaa?gacaacaagg?gcctctcttt?ccggcttgcc?atcaagagaa?agggcgccta 3300

ggaatttttg?agtctggaat?gtcgtggggt?ctggaagagg?ctattcctcc?ccggatggaa 3360

cgcccaagcc?ccacccacgg?catgccttcc?cacccgtaca?gtttcccagc?ccttagggtt 3420

tgccggcctt?aacccccttg?gggaggccac?cgaggtgcag?agagggcggg?tgccagaggg 3480

aaaggggaag?aagagtgtgc?cctgggttcc?caggtggaga?aattctgagg?tctcttccca 3540

gcgcagtgcc?ctggcagcga?agctctgcca?ggttgctcgg?attgatcccc?tggcgggatg 3600

cacgagggct?gggggtgtcc?ttaggtcccg?ggggagggag?ggcgctcgga?ccagtgaggc 3660

ttcctgcctg?acacgctgcg?catctccacg?gccttgctga?tcttgcagaa?acccgac 3717

<210>2

<211>23

<212>DNA

＜213〉artificial sequence

<400>2

ggataaccgt?attaccgcca?tgc 23

<210>3

<211>26

<212>DNA

＜213〉artificial sequence

<400>3

gctctagaca?aaacaaactc?ccattg 26

<210>4

<211>31

<212>DNA

＜213〉artificial sequence

<400>4

gggatgatag?ataatagttg?ccagtgaagc?c 31

<210>5

<211>25

<212>DNA

＜213〉artificial sequence

<400>5

gtcgggtttc?tgcaagatca?gcaag 25

<210>6

<211>38

<212>DNA

＜213〉artificial sequence

<400>6

ccctcgaggg?atgatagata?atagttgcca?gtgaagcc 38

<210>7

<211>32

<212>DNA

＜213〉artificial sequence

<400>7

ggaattcgtc?gggtttctgc?aagatcagca?ag 32

<210>8

<211>22

<212>DNA

＜213〉artificial sequence

<400>8

gaagtaggga?tacagtgtgg?cg 22

<210>9

<211>22

<212>DNA

＜213〉artificial sequence

<400>9

gggagggaag?cgaggcttct?ct 22

<210>10

<211>467

<212>DNA

＜213〉artificial sequence

<400>10

gaagtaggga?tacagtgtgg?cggcaccttc?cagaggcccc?gggcgcaggg?tagaccgggg 60

cggggcggcc?cgcggacagg?tgcagcccca?ggcgcaggcg?cactcgcgcc?tcccggcgca 120

ggcggtgaac?ctcgccccac?cccagcccct?ccggggggca?gctgggccgg?gtcgggaggg 180

gcccaccagc?ccgggagaca?ctccatatac?ggccaggccc?gctttacctg?ggctccggcc 240

aggccgctcc?ttctttggtc?agcacagggg?acccgggcgg?gggcccaggc?cgctaacccg 300

ccgggggagg?gggctccagt?gcccaacacc?caaatatggc?tcgagaaggg?gagcgacatt 360

ccagtgaggc?ggctcggggg?gagaacccgc?gggctatata?aaacctgagc?gtggggacca 420

gcggccaccg?cagcggacag?cgccgagaga?agcctcgctt?ccctccc 467

Claims (4)

1. skeletal muscle specificity promoter, it is characterized in that: the gene order of this promotor is shown in Seq ID No:1.

2. a recombinant nucleic acid sequence is characterized in that: include promotor or this promoter variants or the fragment of gene order shown in Seq ID No:1 in the sequence.

3. according to the recombinant nucleic acid sequence under the claim 2, it is characterized in that: also include at least a functional gene in the sequence, described functional gene comprises raising meat quality gene or accelerates speed of growth gene or disease-resistant gene or the gene of the raising price of deed or gene or its combination of production certain drug.

4. an expression system is characterized in that: include as claim 2 or 3 described recombinant nucleic acid sequences.

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