CN104774838A - Bovin CMYA1 muscle specific expression core promoter and preparation method thereof - Google Patents

Abstract

The invention discloses a bovin CMYA1 muscle specific expression core promoter and a preparation method thereof. The preparation method comprises the following steps: firstly cloning a full-length fragment 1155bp of the bovin CMYA1 muscle specific expression core promoter, building a pEGFP-1-CMYA1 promoter carrier, and transfecting mice C2C12 myoblast, bovine skeletal muscle satellite cells and bovine fibroblast to prove the muscle specificity expression of the carrier; then, carrying out the deficiency research on CMYA1 promoter, totally designing 8 deletion fragments of the CMYA1 promoter, respectively building deletion fragment carriers of pGL3-basic-CMYA1 promoters, and transfecting C2C12 cells; and finally verifying, thereby obtaining the CMYA1 core promoter of which the gene sequence is SQ2. The CMYA1 promoter is specifically expressed in muscle cells, and the promotion activity is high; moreover, such the characteristic can be utilized by people to make a research on the development and differentiation of muscles.

Description

A kind of ox CMYA1 muscle specific expresses core promoter and preparation method

Technical field

The invention belongs to the gene engineering technology field in biological technical field, particularly a kind of ox CMYA1 muscle specific expresses core promoter and preparation method.

Background technology

1, CMYA1 progression, different plant species CMYAl (Cardiomyopathy associated protein 1, cardiomyopathies associated protein 1) all contain the structural domain of 5 high conservatives, be respectively the region of proline rich, 16 amino acid whose tumor-necrosis factor glycoproteinss (being again Xin tumor-necrosis factor glycoproteins), DNA binding domains, SH3 structural motif and nuclear localization signals.Ox CMYA1 gene (CMYA1Gene ID:509670) is positioned at Chromosome 22:12549676 ~ 12558895bp, there is two exons intron, to encode 1820 amino acid, coding region total length 6272bp, the isoelectric points of proteins (Isoelectricpoint) derived: 6.4162, molecular weight (Molecular weight): 194814.91g/mol.

CMYA1 gene is found in the coronary sulcus of chicken embryo by mRNA differential display technique the earliest, again called after XIRPl or Xin gene.The adhesion joint of CMYA1 protein localization in intercalated disc and the tendon binding site of skeletal muscle, there are two exons in CMYA1 gene in Mammals, and second exon comprises whole proteins encoded region (Julia Otten et al., 2010).The aminoacid sequence of chicken (cXin) and mouse (mXin) has the homology of 46%, contains proline(Pro) enrich district, 16 amino acid repeating units, nucleic acid signal for locating and SH3 binding motif through prediction skeletal muscle specificity CMYA1 gene.Expression pattern analysis display CMYA1 gene is expressed in voluntary muscle.Then successfully obtaining by building mouse mXin β targeting vector the mXin β depleted mice model isozygotied, finding that the mouse core muscle layer lacking mXin β gene is completely unordered, cardiac shape abnormal, interventricular septum disappearance, cardiac diastolic function are disorderly, serious growth retardation and post-natal lethality.MXin β plays the function of necessity in postnatal cardiac growth and animals survived regulating N-cadherin (N-cadherin) to control, confirm that CMYA1 gene forms developmental vital role at heart further.Xin mRNA expresses promotion in the muscle tissue of 12 hours after muscle injury, and immunohistochemical analysis proves that Xin gene is expressed in muscle satellite cell.Myogenetic differentiation factor MyoD, the raw factor M yf-5 of flesh and the raw enhancement factor MEF2 of flesh of transcribing has the ability to activate Xin.A lot of research shows, CMYA1 specific gene expression is in muscle tissue, thus CMYA1 promotor is probably that muscle specific is expressed, and find no the promotor of people to ox CMYA1 gene and study, the utilization for follow-up CMYA1 muscle specific promoter lays the foundation by the present invention.

2, muscle specific expresses promotor, promotor is positioned at the section of DNA sequence near gene 5 ' end upstream transcription initiation site, according to the difference of genetic expression attribute, promotor can be divided into constitutive promoter and specific promoter, the former in a organized way in all can express by promotor gene, the latter only in the particular organization of certain developmental stage promotor gene express.In recent years, people have been separated many muscle specific promoter, as the promotor of the muscle specific genes such as muscle creatine kinase (MCK), skeletal muscle α-actin, myoglobulin heavy chain (MyHC), myogenic regulatory factor family (MyoD, Myf5, Mrf4 and Myogenin), and then these promotors are furtherd investigate the propagation of muscle cell, Differentiation mechanism and transcriptional activity.Kind, the quantity of the upstream region transcriptional regulatory element of promotor, putting in order has a very important role to the transcriptional activity of tissue-specific gene.Containing multiple upstream transcription controlling element in muscle specific promoter, mainly contain: M-CAT element, MEF-1 element, MEF-2 element, Trex/MEF-3 element, CACCC box, SRE element, MPEX element, Pax3/7 element.

Domestic also have some investigator's separating clones promotor of some genes, as clone and the checking of mouse MCK promotor, clone obtains mouse MCK promotor 1355bp, do not do disappearance screening core promoter fragment, also there is investigator to clone and obtain goat MyoG gene promoter 1200bp, do not carry out promoter deletion research, obtain core promoter fragment yet.Although some muscle specific promoter obtain, its activity differs with efficiency variance, and fragment length is also different.A lot of research shows, the core controlling element of promotor is distributed in 3 ' end non-translational region of promotor, the present invention takes this as a foundation, fix 3 ' end, hold in promotor 5 ' and do disappearance checking, in the hope of finally obtaining shorter CMYA1 muscle specific core promoter sequence, facilitate the utilization of this muscle specific promoter.

The present invention has carried out comprehensive analysis to CMYA1 gene and promoter region thereof, consider the position characteristics of promotor in genome, find in CMYA1 gene First Exon first alkali yl upstream 1135bp and 20bp region, downstream, not containing conserved sequence " TATA " frame and " CAAT " box, containing conserved sequence " GC " box, express transcriptional regulatory element SRE element containing muscle specific, MEF-1 element (E-box), MEF-2 element, TreX element and Pax3/7 element.The core sequence of SRE element is 5 '-CC [A/T] GG-3 ', i.e. serum response element, SRE element is all there is in the promotor of known α-actin, myosin light chain (MyLC), myodystrophy gene (dystrophin) and early growth response factors-1 (early growth response-1, Egr-1).The core sequence of MEF-1 element (E-box) is 5 '-CANNTG-3 ', it is mainly present in myogenic regulation and control family factor M yoD, Myf5, Myogenin and MRF4 gene, and they play vital effect in Skeletal Muscle Growth and growth course.MEF-2 element, its core sequence is 5 '-[C/T] TAAAAAT-AAC [C/T]-3 ', and this element is present in the promotor enhanser region of myosin light chain 2A gene.The MEF-2 cis-acting elements being rich in A/T motif can be combined with the MEF2 factor, and the MEF2 factor is MADS-box superfamily albumen a member, is the important Downstream regulatory factor of Mammals myogenic regulatory gene MyoD.TreX binding sequence is 5 '-[G/C/T] [A/G/C] NGAT [A/G] G [G/C/T] [G/C/T]-3 ', and TreX element is proved to be most important to the expression of MCK gene promoter in skeletal muscle and cardiac muscle.Pax3/7 element, its core sequence is 5 '-GTAAC-3 ', is combined with trans-acting factor Pax3 and Pax7 mixture.Pax3/7 element is mainly present in the promotor upstream regulatory region of myogenic regulatory factor MRF gene, and the expression of initial MRF gene, plays key effect in the skeletal development of embryo.Therefore, the CMYA1 promotor that the present invention obtains, its feature is as follows: full length fragment 1155bp, is positioned at CMYA1 gene First Exon first alkali yl upstream 1135bp and downstream 20bp, not containing conserved sequence " TATA " frame and " CAAT " box, containing conserved sequence " GC " box.

Summary of the invention

The object of the invention is for the deficiencies in the prior art, and propose a kind of ox CMYA1 muscle specific expression core promoter and preparation method.

The present invention solves its technical problem and takes following technical scheme to realize:

A kind of ox CMYA1 muscle specific expresses core promoter, and the gene order of this core promoter is SQ2.

Ox CMYA1 muscle specific expresses a preparation method for core promoter, comprises step as follows:

(1) clone of ox CMYA1 muscle specific promoter,

On NCBI, comparison ox CMYA1 complete genome sequence is in chromosomal position, and find First Exon, according to the position characteristics of promotor in gene order, clone First Exon first alkali yl upstream 1135bp and downstream 20bp sequence, obtain the promoter gene sequence SQ1 of 1155bp altogether;

(2) build pEGFP-1-CMYA1 promoter vector and verify that above-mentioned promotor is only specific expressed in muscle cell,

1. be designed for the PCR upstream and downstream primer of this fragment of amplification, upstream primer 5 ' is held and is added restriction enzyme site XhoI (CTCGAG), and protection base is CCG, downstream primer 5 ' is held and is added restriction enzyme site HindIII, (AAGCTT), protection base is CCC, and pcr amplification goes out this fragment;

2. then build pEGFP-1-CMYA1 promoter vector, transfection C2C12 cell, bovine muscle satellite cell and bovine fibroblasts, verify this promotor only specific expressed in muscle cell respectively;

(3) core promoter is screened,

1. first carry out the design of promoter deletion fragment, design deletion-primers 8 altogether right, upstream primer 5 ' is held and is added restriction enzyme site XhoI, downstream primer 5 ' is held and is added restriction enzyme site HindIII, amplifies corresponding promoter deletion fragment, and difference called after P1, P2, P3, P4, P5, P6, P7 and P8, carrier construction pGL3-Basic-CMYA1 promoter deletion fragment vector respectively, with pRL-TK carrier cotransfection C2C12 cell respectively, pGL3-Basic is negative control, and pGL3-Control is positive control;

2. transfection is after 24 hours, cracking C2C12 cell, the dual-luciferase reporter system detection kit of producing with PROMEGA company detects CMYA1 promoter activity, filters out CMYA1 core promoter, this core promoter sequence length 477bp, gene order is SQ2;

3. use C2C12 cell and bovine muscle satellite cell transfection experiment, prove that this core promoter still maintains its character specific expressed in muscle cell;

And, the 8 pairs of deletion-primers 1. in step of described step (3) screening core promoter as shown in Table 1 below,

The design of table 1.CMYA1 promoter deletion fragment primer

Advantage of the present invention and positively effect are:

1. CMYA1 promotor of the present invention is specific expressed in muscle cell, and it is very high to start activity, people can utilize the Development And Differentiation of this characteristic to muscle to study, and a certain protein of some gene specifically expressing in muscle tissue or hormone etc. can be made, be conducive to the improvement of the prevention of disease, diagnosis, treatment and domestic animal Meat Quality.

2. a lot of specificity promoter starts expression in the myocyte of end differentiation eventually, and promotor of the present invention does not find similar situation.

3. the muscle specific promoter core fragment that the present invention obtains only has 477bp, shorter than other muscle specific promoter fragment a lot, and applicability is stronger, and is rich in muscle specific promoter transcriptional regulatory element.

Accompanying drawing explanation

Fig. 1 is pEGFP-1 carrier schematic diagram;

Fig. 2,3 and 4 is respectively pEGFP-1-CMYA1 promotor (1155bp) recombinant vectors transfection C2C12 cell, bovine muscle satellite cell and bovine fibroblasts figure respectively;

Fig. 5 is promoter deletion fragment figure;

Fig. 6 is pGL3-basic carrier schematic diagram;

Fig. 7 and 8 is respectively structure promoter deletion fragment vector bacterium liquid PCR and enzyme cuts qualification figure;

Fig. 9 is pRL-TK carrier schematic diagram;

Figure 10 is luciferase reporter gene detected result figure;

Figure 11,12 and 13 is pEGFP-CMYA1 core promoter (477bp) recombinant vectors transfection C2C12 cell, bovine muscle satellite cell and bovine fibroblasts figure respectively;

Figure 14 is the structural representation of ox muscle specific expression vector pCMYA1-DGAT1;

Figure 15 is the expression amount schematic diagram of quantitative PCR detection DGAT1.

Embodiment

Be further described the invention process below in conjunction with accompanying drawing, following examples are descriptive, are not determinate, can not limit protection scope of the present invention with this.

A kind of ox CMYA1 muscle specific expresses core promoter and preparation method

The experimental technique used in following embodiment if no special instructions, is ordinary method; The material used, reagent etc., if no special instructions, all can obtain from commercial channels.It is as follows that the preparation method that ox CMYA1 muscle specific of the present invention expresses core promoter comprises step:

1, the clone of ox CMYA1 muscle specific promoter and checking

Concrete steps operation is as follows:

(1) clone of ox CMYA1 muscle specific promoter

On NCBI, comparison ox CMYA1 complete genome sequence (CMYA1 Gene ID:509670) is in chromosomal position, and find First Exon, according to the position characteristics of promotor in gene order, by analysis in conjunction with the controlling element feature that muscle specific promoter comprises, clone First Exon upstream 1135bp and downstream 20bp sequence, 1155bp promoter sequence altogether, concrete design of primers is as follows:

Design CMYA1 promotor upstream primer F-CCG cTCGAGgGCCAAT GCTGCCTGCCTGA (underscore place is XhoI restriction enzyme site), downstream primer R-CCC aAGCTTgCTGCCCTAGCTCGGGGTCT (underscore place is HindIII restriction enzyme site), with Simmental Crossbred Progeny genomic dna for template.

PCR reaction system: adopt 20 μ L reaction systems, 0.1 μ L Taq DNA Polymerase (5U/ μ L), 2 μ L 10 × TaqPCR buffer, 0.4 μ L dNTPs, 1 μ L template, upstream primer 0.8 μ L (10 μMs), downstream primer 0.8 μ L (10 μMs), 14.9 μ L sterilizing ultrapure waters.

PCR reaction conditions is, 95 DEG C of denaturation 3min, 95 DEG C of sex change 30s, and 65 DEG C of annealing 30s, 72 DEG C extend 50s, 30 circulations, last 72 DEG C of 5min.

Get 5 μ L amplified productions, agarose gel electrophoresis detects.The method reclaiming test kit according to sepharose DNA reclaims 1155bp CMYA1 promoter gene fragment.Be connected into pUCm-T carrier according to the method for T-carrier PCR primer Cloning Kit, obtain carrier pUCm-T-CMYA1 promotor, transformation of E. coli DH5 α, order-checking qualification, successful clone obtains CMYA1 promotor 1155bp, and obtaining gene order is SQ1.

SQ1:

ggccaatgctgcctgcctgacgctgactcaatggaaaagagtgagcagtgcctggcatcacttcctggcaccagagggcacccaggatgtcagaagaaagaccctgttctgccagctagcggaacacaacttcgacatggaacctgggcaaaatgcctcagtcatttgtgctctgcatggtctcctgatgcccctcgccacactgcccttcccagcctaaatgccaggatctctggagcctactaaggcccttgggctctgggcagccaaggaggggcagccgtgacctccacacacaaacatacacacatactccttctgtgacgggaagctgatgactccgggtcagaaataacccccgagtcagctccccgcgtgctgcccctcccctctcctccgttcccagaaagcctgggccctgagtcagaggcgagagcctgacagtaaacaagacccagataatctgctgtgaacaccgcagaccatgctggggaatcctctgacaatcccggacccggtgaagaaccggggcctggaatatgtcttcccccaaacccagctgtgtgcactcagggctgtatatacctgtgcggtgggatgcggggagggatggaagagacaggggaaaggtctagcgcaggagtggggagaagaagggccttactctcaggggtatccgggctgggaaatgaggagcaagggcctaggcctgccctcagggggcctccagtctcctggcaggatgggcacacgccacacgtcagacatagtcagaggctggggtagagacgtgggggctcacaggatggggggccaagttgaccaggtgtcagatgctcctccttattcattccctcagaatagtctgggatgtccgcaccccacaccacagctgagtcactgaccccaatcctcagaggagacttggatgaatgaagctctcccccactcactcctcttcccctccctagcagaaaggacatgtaactgatgcccgagatggagagtaacttccagagccctaaaaataactgaaccacttcatactctgaccttagcctcccccactgcccccacctcggcccaccccctaaccagccagataaaggcagctgctgaggctggcaggggacacagaccccgagctagggcagc

(2) pEGFP-1-CMYA1 promoter vector is built

XhoI and HindIII double digestion carrier pUCm-T-CMYA1 promotor:

Enzyme cuts system: adopt 50 μ L reaction systems, ultrapure water 21 μ L, 10 × Fast Digest Buffer 5 μ L, pUCm-T-CMYA1 promotor 20 μ L, Fast Digest XhoI 2 μ L, Fast Digest HindIII 2 μ L.

Reaction conditions is, 37 DEG C of enzymes cut 1h, 80 DEG C of enzyme deactivation 10min.Glue reclaims digestion products.

XhoI and HindIII double digestion carrier pEGFP-1, as shown in Figure 1,

Enzyme cuts system: adopt 50 μ L reaction systems, ultrapure water 21 μ L, 10 × Fast Digest Buffer 5 μ L, pEGFP-1 20 μ L, Fast Digest XhoI 2 μ L, Fast Digest HindIII 2 μ L.

Reaction conditions: 37 DEG C of enzymes cut 1h, 80 DEG C of enzyme deactivation 10min.Glue reclaims digestion products.

Linked system: connect employing 20 μ L reaction system, pUCm-T-CMYA1 promotor reclaims product 2 μ L, and pEGFP-1 reclaims product 4 μ L, T ₄dNA Ligase 0.5 μ L, T ₄dNA Ligase Buffer 2 μ L, ultrapure water 11.5 μ L.Reaction conditions is 22 DEG C and connects 4h.Transformation of E. coli DH5 α, enzyme cuts qualification, and order-checking qualification, obtains carrier pEGFP-1-CMYA1 promotor.

(3) CMYA1 promoter activity and specificity verification

Cell cultures: this cultured cells is mouse C2C12 cell, bovine muscle satellite cell and bovine fibroblasts, cell culture medium configures: the high sugar of 79%DMEM+20% foetal calf serum+1% mycillin.

Cell dissociation: clean cell 1-2 time with PBS, add 3mL 0.25% trypsinase-EDTA Digestive system, 37 DEG C of digestion 1-2 minute, collecting cell Digestive system, the centrifugal 5min of 1000rpm.

Cell transfecting: C2C12 cell, bovine muscle satellite cell and bovine fibroblasts are cultivated in 35mm culture dish at day before transfection, makes second day cell density can reach Tissue Culture Dish and is about 70-80%.Before carrying out following transfection procedure, changing fresh cell culture fluid in 35mm into.The volume of nutrient solution is about 1.75mL.

Lipofectamine2000 reagent is mixed gently, 3.5 μ Llipofectamine2000 are added successively in a cleaning sterile centrifuge tube, appropriate DMEM solution and the 1.75 μ g plasmid pEGFP-1-CMYA1 promotors not containing microbiotic and glutamine, make final volume be 100 μ L, blow and beat mixing gently with rifle.

Hatch 15 minutes for 25 DEG C.100 μ L lipofectamine 2000-plasmid pEGFP-1-CMYA1 mixtures are all added in culture dish.The nutrient solution containing lipofectamine2000-plasmid pEGFP-1-CMYA1 is absorbed after 6 hours.Add 2ml fresh cell medium to continue to cultivate.

Transfection is after 24 hours, at fluorescence microscopy Microscopic observation mouse C2C12 cell, as shown in Figure 2, bovine muscle satellite cell, as shown in Figure 3, and bovine fibroblasts, as shown in Figure 4, egfp expression situation, finds that C1C12 cell and bovine muscle satellite cell have egfp expression, bovine fibroblasts does not have egfp expression, proves that CMYA1 promotor is that muscle specific is expressed.

2, ox CMYA1 muscle specifics express the screening of promotor core promoter

(1) CMYA1 promoter deletion gene fragment is cloned

First be template according to CMYA1 promotor full length sequence, design deletion-primers 8 is right, and as shown in table 1 and Fig. 5, upstream and downstream primer 5 ' end adds restriction enzyme site XhoI and HindIII respectively.8 pairs of primers are utilized to obtain corresponding 8 promoter deletion fragments respectively, and difference called after P1, P2, P3, P4, P5, P6, P7 and P8.

The design of table 1.CMYA1 promoter deletion fragment primer

PCR reaction system: all adopt 20 μ L reaction systems, 0.1 μ L Taq DNA Polymerase (5U/ μ L), 2 μ L10 × Taq PCR buffer, 0.4 μ L dNTPs, 1 μ L template, upstream primer 0.8 μ L (10 μMs), downstream primer 0.8 μ L (10 μMs), 14.9 μ L sterilizing ultrapure waters.

PCR reaction conditions is, 95 DEG C of denaturation 3min, 95 DEG C of sex change 30s, and 62 DEG C of annealing 30s, 72 DEG C extend 50s, 30 circulations, last 72 DEG C of 5min.

All get 5 μ L amplified productions, agarose gel electrophoresis detects.The method reclaiming test kit according to sepharose DNA reclaims 8 sections of CMYA1 promoter gene fragments.Be connected into pUCm-T carrier respectively according to the method for T-carrier PCR primer Cloning Kit, obtain pUCm-T-CMYA1 promoter deletion fragment vector, transformation of E. coli DH5 α, order-checking qualification.

(2) pGL3-basic-CMYA1 promoter deletion fragment vector is built

Be connected respectively on pGL3-basic carrier by the promoter fragment P1-P8 that 8 lack, as shown in Figure 6, its specific embodiment is as follows for carrier:

XhoI and HindIII double digestion carrier pUCm-T-CMYA1 promoter deletion fragment:

Enzyme cuts system: adopt 50 μ L reaction systems, ultrapure water 21 μ L, 10 × Fast Digest Buffer 5 μ L, pUCm-T-CMYA1 promoter deletion fragment vector 20 μ L, Fast Digest XhoI 2 μ L, Fast Digest HindIII 2 μ L.

Reaction conditions: 37 DEG C of enzymes cut 1h, 80 DEG C of enzyme deactivation 10min.Glue reclaims digestion products.

XhoI and HindIII double digestion carrier pGL3-basic:

Enzyme cuts system: adopt 50 μ L reaction systems, ultrapure water 21 μ L, 10 × Fast Digest Buffer 5 μ L, pGL3-basic20 μ L, Fast Digest XhoI 2 μ L, Fast Digest HindIII 2 μ L.

Reaction conditions: 37 DEG C of enzymes cut 1h, 80 DEG C of enzyme deactivation 10min.Glue reclaims digestion products.

Linked system: connect employing 20 μ L reaction system, pUCm-T-CMYA1 promoter deletion fragment reclaims product 2 μ L, and pGL3-basic reclaims product 4 μ L, T ₄dNA Ligase 0.5 μ L, T ₄dNA Ligase Buffer 2 μ L, ultrapure water 11.5 μ L.Reaction conditions is 22 DEG C and connects 4h.Transformation of E. coli DH5 α, bacterium liquid PCR and enzyme cut qualification, and as shown in Figure 7, Figure 8, order-checking qualification, obtains pGL3-basic-CMYA1 promoter deletion fragment vector 8.

(3) the activity checking of ox CMYA1 promoter deletion fragment

The dual-luciferase reporter system checking promoter deletion fragment activity that this checking adopts PROMEGA company to produce, concrete operations are as follows:

Cell cultures: this cultured cells is mouse C2C12 cell, the high sugar of cell culture medium: 79%DMEM+20% foetal calf serum+1% mycillin.

Cell dissociation: treat that C2C12 cell density can reach 35mm Tissue Culture Dish and be about 70-80%, cleans cell 1-2 time with PBS, adds 3mL 0.25% trypsinase-EDTA Digestive system, 37 DEG C of digestion 1-2 minute, collecting cell Digestive system, the centrifugal 5min of 1000rpm.

Cell transfecting: this adopts the mode of electrotransfection to carry out cell transfecting, the ECM 2001 multifunction electric fusion instrument that electrotransfection adopts BTX to produce, concrete electricity turns parameter: voltage: 240V, the electric shock time: 4ms, concrete operations: with electrotransfection damping fluid suspension cell, plasmid pGL3-basic-CMYA1 promoter deletion fragment of plasmid and pRL-TK plasmid, as shown in Figure 9, 20ug is added in cell suspension altogether, pGL3-basic-CMYA1 promoter deletion fragment of plasmid and pRL-TK plasmid concentration are than being 30:1, be placed in precooling 10min on ice, cell suspension is transferred in electric shock cup, perform electric shock, after electric shock terminates, in continuing precooling 10min on ice, neutralize with 2ml cell culture complete medium, cultivate in 35mm culture dish.

Luciferase reporter gene detects: after cell transfecting 24h, cracking C2C12 cell, cell pyrolysis liquid is used for the luciferase reporter gene detection kit detection that PROMEGA company produces, finally, by being activated after fluorescent value that the Photinus pyralis LUC that starts and express produces through catalysis and the fluorescent value that the catalysis of positive control renilla luciferase produces proofread by CMYA1 promotor, draw ox CMYA1 muscle specific promoter core fragment (P7), this fragment length 477bp, gene order is SQ2.

SQ2:

gggctgggaaatgaggagcaagggcctaggcctgccctcagggggcctccagtctcctggcaggatgggcacacgccacacgtcagacatagtcagaggctggggtagagacgtgggggctcacaggatggggggccaagttgaccaggtgtcagatgctcctccttattcattccctcagaatagtctgggatgtccgcaccccacaccacagctgagtcactgaccccaatcctcagaggagacttggatgaatgaagctctcccccactcactcctcttcccctccctagcagaaaggacatgtaactgatgcccgagatggagagtaacttccagagccctaaaaataactgaaccacttcatactctgaccttagcctcccccactgcccccacctcggcccaccccctaaccagccagataaaggcagctgctgaggctggcaggggacacagaccccgagctagggcagc

Concrete operations are as follows:

96 hole blackboards are adopted to carry out Dual-Luciferase detection, first every hole adds the cell pyrolysis liquid of 100ul, then LARII solution is added, values of chemiluminescence detection is carried out rapidly by multi-functional microplate reader, what now detect is Photinus pyralis LUC activity, then Stop and Glo reagent is added, abundant mixing, carry out values of chemiluminescence detection rapidly, what now detect is renilla luciferase activity, the activity of CMYA1 promotor is obtained by the calibration values of two kinds of enzymes, as shown in Figure 10, as seen from the figure, P7 promoter fragment relative fluorescence is higher, and fragment is shorter, for CMYA1 core promoter.

(4) CMYA1 core promoter muscle specific expresses checking

Build pEGFP-CMYA1 core promoter (477bp) carrier, and difference transfection C2C12 cell, as Figure 11, bovine muscle satellite cell, as Figure 12 and bovine fibroblasts, as Figure 13, find that CMYA1 core promoter is still only specific expressed in C2C12 cell and bovine muscle satellite cell, and in bovine fibroblasts, have no expression, illustrate that CMYA1 core promoter (477bp) is not only very well active, and still maintain its character specific expressed in muscle cell.Concrete system and operation are with the clone of ox CMYA1 muscle specific promoter and checking.

(5) embodiment

The experimental technique used in following embodiment if no special instructions, is ordinary method; The material used, reagent etc., if no special instructions, all can obtain from commercial channels.

PcDNA 3.1 plasmid vector is transformed, utilizes Mlu I and Hind III double digestion to remove CMV promoter, insert CMYA1 core promoter (477bp).Eco R I and Eco RV restriction enzyme site is utilized by ox DGAT1 gene (NM_174693.2) CDS sequence SQ3 to insert the multiple clone site of pcDNA 3.1 plasmid vector.Obtain ox muscle specific expression vector pCMYA1-DGAT1, as shown in figure 14.

Wherein, SQ3:

ATGGGCGACCGCGGCGGCGCGGGCGGCTCCCGGCGCCGGAGGACGGGGTCGCGGCCTTCGATCCAGGGCGGCAGTGGGCCCGCGGCAGCGGAAGAGGAGGTGCGGGATGTGGGCGCTGGAGGGGACGCGCCGGTCCGGGACACAGACAAGGACGGAGACGTAGACGTGGGCAGCGGCCACTGGGACCTGAGGTGTCACCGCCTGCAGGATTCCCTGTTCAGTTCTGACAGTGGCTTCAGCAACTACCGTGGCATCCTGAATTGGTGTGTGGTGATGCTGATCTTAAGCAACGCACGGTTATTTCTAGAGAACCTCATCAAGTATGGCATCCTGGTGGACCCCATCCAGGTGGTGTCTCTGTTCCTGAAGGACCCCTACAGCTGGCCAGCTCTGTGCCTGGTCATTGTGGCCAATATCTTTGCCGTGGCTGCGTTCCAGGTGGAGAAGCGCCTGGCCGTGGGAGCTCTGACGGAGCAGGCGGGGCTGCTGCTGCACGGGGTCAACCTGGCCACCATTCTCTGCTTCCCAGCGGCCGTGGCCTTTCTCCTCGAGTCTATCACTCCAGTGGGCTCCGTGCTGGCCCTGATGGTCTACACCATCCTCTTCCTCAAGCTGTTCTCCTACCGGGACGTCAACCTCTGGTGCCGAGAGCGCAGGGCTGGGGCCAAGGCCAAGGCTGCTTTGGCAGGTAAGGCGGCCAACGGGGGAGCTGCCCAGCGCACCGTGAGCTACCCCGACAACCTGACCTACCGCGATCTCTACTACTTCCTCTTCGCCCCCACCCTGTGCTACGAGCTCAACTTCCCCCGCTCCCCCCGCATCCGAAAGCGCTTCCTGCTGCGGCGACTCCTGGAGATGCTGTTCCTCACCCAGCTCCAGGTGGGGCTGATCCAGCAGTGGATGGTCCCGGCCATCCAGAACTCCATGAAGCCCTTCAAGGACATGGACTACTCCCGCATCGTGGAGCGCCTCCTGAAGCTGGCGGTCCCCAACCACCTCATCTGGCTCATCTTCTTCTACTGGCTCTTCCACTCCTGCCTGAACGCCGTGGCTGAGCTCATGCAGTTTGGAGACCGCGAGTTCTACCGGGACTGGTGGAACTCCGAGTCCATCACCTACTTCTGGCAGAACTGGAACATCCCTGTTCACAAGTGGTGCATCAGACACTTCTACAAGCCCATGCTCCGGCGGGGCAGCAGCAAGTGGGCAGCCAGGACGGCAGTGTTTCTGGCCTCCGCCTTCTTCCACGAGTACCTGGTGAGCATCCCCCTGCGCATGTTCCGCCTCTGGGCCTTCACCGGCATGATGGCGCAGATCCCGCTGGCCTGGATAGTGGGCCGCTTCTTCCGCGGCAACTACGGCAACGCGGCCGTGTGGCTGTCACTCATCATCGGGCAGCCGGTGGCCGTCCTGATGTACGTCCACGACTACTACGTGCTCAACCGTGAGGCGCCGGCAGCCGGCACCTGA

Recombinant expression vector pCMYA1-DGAT1 transfection C2C12 cell, transfection group and untransfected control group arrange 3 repetitions respectively, extract RNA after transfection 48h, adopt the expression amount of quantitative PCR detection DGAT1.Experimental result is shown in Figure 15, quantitative PCR detection result shows, after transfection pCMYA1-DGAT1, the expression amount of DGAT1 gene in C2C12 cell is 30.8 times of control group, prove that CMYA-1 promotor successfully orders about the DGAT1 of external source at C2C12 cells, and difference is between the two extremely remarkable compared with untransfected group.This example demonstrated the CMYA1 core promoter (477bp) that we filter out and there is good myogenic expression specificity, and shorter than other muscle specific promoter fragment a lot, and applicability is stronger.

Claims (3)

1. ox CMYA1 muscle specific expresses a core promoter, it is characterized in that: the gene order of this core promoter is SQ2.

2. ox CMYA1 muscle specific expresses a preparation method for core promoter, it is characterized in that: comprise step as follows:

(1) clone of ox CMYA1 muscle specific promoter,

On NCBI, comparison ox CMYA1 complete genome sequence is in chromosomal position, and find First Exon, according to the position characteristics of promotor in gene order, clone First Exon first alkali yl upstream 1135bp and downstream 20bp sequence, obtain the promoter gene sequence SQ1 of 1155bp altogether;

(2) build pEGFP-1-CMYA1 promoter vector and verify that above-mentioned promotor is only specific expressed in muscle cell,

1. be designed for the PCR upstream and downstream primer of this fragment of amplification, upstream primer 5 ' is held and is added restriction enzyme site XhoI (CTCGAG), and protection base is CCG, downstream primer 5 ' is held and is added restriction enzyme site HindIII, (AAGCTT), protection base is CCC, and pcr amplification goes out this fragment;

2. then build pEGFP-1-CMYA1 promoter vector, transfection C2C12 cell, bovine muscle satellite cell and bovine fibroblasts, verify this promotor only specific expressed in muscle cell respectively;

(3) core promoter is screened,

1. first carry out the design of promoter deletion fragment, design deletion-primers 8 altogether right, upstream primer 5 ' is held and is added restriction enzyme site XhoI, downstream primer 5 ' is held and is added restriction enzyme site HindIII, amplifies corresponding promoter deletion fragment, and difference called after P1, P2, P3, P4, P5, P6, P7 and P8, carrier construction pGL3-Basic-CMYA1 promoter deletion fragment vector respectively, with pRL-TK carrier cotransfection C2C12 cell respectively, pGL3-Basic is negative control, and pGL3-Control is positive control;

2. transfection is after 24 hours, cracking C2C12 cell, the dual-luciferase reporter system detection kit of producing with PROMEGA company detects CMYA1 promoter activity, filters out CMYA1 core promoter, this core promoter sequence length 477bp, gene order is SQ2;

3. use C2C12 cell and bovine muscle satellite cell transfection experiment, prove that this core promoter still maintains its character specific expressed in muscle cell.

3. ox CMYA1 muscle specific according to claim 2 expresses the preparation method of core promoter, it is characterized in that: the 8 pairs of deletion-primers 1. in step of described step (3) screening core promoter as shown in Table 1 below,

The design of table 1.CMYA1 promoter deletion fragment primer