CN105238790B - A kind of regulation and control pig AEBP1 promoter and construction method, transfection carrier and construction method, application - Google Patents
A kind of regulation and control pig AEBP1 promoter and construction method, transfection carrier and construction method, application Download PDFInfo
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Abstract
The invention discloses a kind of regulation and control pig AEBP1 promoter and construction method, transfection carrier and construction method, application, the promoter is the deletion fragment of pig AEBP1 gene promoters.The present invention is studied pig AEBP1 promoter first, and by lacking the different fragments of the gene promoter, obtaining 3 kinds has more strongly active new promoter.The new promoter of the present invention can regulate and control the expression of pig AEBP1 genes, and new instrument and selection are provided for swine improvement.Biological experiment shows, 3 deleted carrier pGL3 basic Q1, pGL3 basic Q2, the pGL3 basic Q3 that the present invention is built have more strongly active, wherein pGL3 basic Q1 active highest in Ren sus domestica cell PK 15.The result of the present invention finally obtains 1483bp promoter fragment (337bp to 1819bp), i.e., the promoter fragment included in the deleted carrier pGL3 basic Q1 that the present invention is built, possesses independent startup function.
Description
Technical field
The present invention relates to the construction method of the promoter of regulation and control pig AEBP1 a kind of and promoter, the transfection containing promoter
The construction method and application of carrier and transfection carrier, belong to animal gene engineering technology field.
Background technology
The expression of higher organism gene thus is had strict time and space by the finely regulating of intraor extracellular environment
Order.For the expression regulation of gene by the horizontal common completion of multistage regulation and control, it is a complexity and orderly process, is mainly wrapped
Before including transcription, transcription, after transcription, translation, five levels after translation, wherein transcriptional level control is the most key.Promoter is to turn
Important regulating and controlling element in record level, it is located at DNA structure gene 5 ' and holds upstream, by regulating and controlling base with transcription factor interaction
The expression (military force, 1999) of cause.Therefore, by the research of the structure, function, binding mode to promoter etc., contribute to
It is best understood from how gene plays its function.
AP2 is the marker gene of Adipocyte Differentiation, and its promoter region is present known to during Adipocyte Differentiation
Two important factor C/EBPa and PPAR γ binding site, be up-regulated expression.In recent years, it is neighbouring in aP2 gene promoters
Enhancer region AE-1 is found that another important transcription factor-adipocyte enhancer binding protein (AEBP1).
AEBP1 is a kind of transcriptional repressor, has protease and DNA binding activity (He et al., 1995) concurrently, is regulation
A member (Muise and Ro, 1999) of property B samples (regulatory B-like) carboxypeptidase family, therefore, AEBP1 has carboxylic
Peptase (carboxypeptidase, CP) activity, express and lower in fat generates.CP is caused to live after CP region mutations in AEBP1
Property missing so that AEBP1 loses suppression transcriptional activity, if otherwise AEBP1 there is CP activity can just suppress to transcribe, therefore
It is (He et al., 1995) of crucial importance that AEBP1 CP activity exercises transcriptional control function for it.Adjust and transcribe on AEBP1
The specific mechanism for suppressing function is unclear, it may be possible to is realized by shearing general transcription factor.Research is found, utilizes ferment
Female two-hybrid system, display AEBP1 have interactions between protein with the subunits of G-protein heterotrimer γ 5.AEBP1 Transcription inhibition function
Adjusted by G γ 5, and G γ 5 inhibitory activity is generated by fat stimulates regulation, therefore, AEBP1 can be with fatty generating process
Effector (Park et al., 1999) as signal factor G γ 5.In addition, research also shows that AEBP1 can adjust mitogen element and live
Change the activity of protein kinase (Mitogen-activated protein kinase, MAPK).
AEBP1 has very important biological action during Adipocyte Differentiation.In mouse 3T3-L1 cell lines
In, AEBP1 expression quantity as the increase in differentiation period gradually reduces, is had no in differentiation latter stage in cell differentiation highest at initial stage
Expression (He et al., 1995).AEBP1 is similar in the expression of human body, and it is thin that studies have shown that in the Initial culture of people makes bone
AEBP1 gene cDNAs can be separated in born of the same parents, but last hardening period then closes expression (Ohno et al., 1996).
AEBP1 has two kinds of different transcripts, is separately encoded 2 kinds of different protein, i.e. AEBP1 and sustainer carboxypeptidase
Class sample albumen (Aortic carboxypeptidase-like protein, ACLP), and ACLP is similar shape in AEBP1 non-core
Isomers, the N-terminal of the albumen more 380 amino acid compared with AEBP1.Two kinds of isoforms are led to by AEBP1 genes respectively
Cross coded by two kinds of mRNA caused by alternative splicing mechanism, its function is also entirely different (Ro et al., 2001).It is in situ miscellaneous
Hand over and show that ACLP is expressed in aortic smooth muscle cell, and do not expressed in Skeletal Muscle Cell, broken up in vascular smooth muscle cells
Middle up-regulated expression (Layne et al., 1998), and AEBP1 in Adipocyte Differentiation then expression lower (He et al.,
1995；Kim et al.,2001).AEBP1 expresses highest in brain, and what is taken second place is white adipose tissue, kidney and heart, in palm fibre
Color adipose tissue, skeletal muscle, small intestine, lung, spleen and testis have expression (Ro et al., 2001).Western hybridization displays
ACLP is distributed mainly in adult rats aortic smooth muscle cell, and is not expressed then in tunica adventitia of artery, the heart, liver, skeletal muscle or kidney
(Layne et al.,1998).Therefore, it is significant to AEBP1 genes, the especially research to AEBP1 promoters.Cut
Only at present, the report of research pig AEBP1 gene promoter subfunctions is still seen.
The content of the invention
The technical problems to be solved by the invention are to provide the promoter of regulation and control pig AEBP1 a kind of and the structure side of promoter
Method, the construction method of transfection carrier and transfection carrier containing promoter and application, the promoter activity is stronger, possesses independent
Startup function.
To achieve these goals, the technical solution adopted in the present invention is to provide a kind of regulation and control pig AEBP1 promoter,
The promoter is the nucleotide sequence shown in SEQ ID NO.10, SEQ ID NO.11 or SEQ ID NO.12.
The technical solution adopted in the present invention also resides in the construction method for providing a kind of regulation and control pig AEBP1 promoter, bag
Include following steps：
(1) with GenBank accession number：CU928907 nucleotides sequence is classified as template, designs forward and reverse primers F/R, and PCR expands
Increase, select amplified fragments First Exon ATG upstreams 2000bp sequences as promoter candidate segment；
(2) using promoter candidate segment as template, design 3 deletion fragments P1, P2, P3 forward and reverse primer P1F/R,
P2F/R, P3F/R, enter performing PCR amplification respectively, obtain pcr amplification product, as regulate and control pig AEBP1 promoter.
Forward and reverse primers F/R is：
Forward primer F：5’-GTGGCTGCCTGTTCATCATCG-3’
Reverse primer R：5’-CTTTGCGGACTGGAAGTGTG-3’.
Forward and reverse primer P1F/R is：
Forward primer P1F：5’-CTAGCTAGCCGAGAAGCCTTATGGAAACA-3’
Reverse primer P1R：5’-CCCAAGCTTCGAACCGCCTTCTCCAAA-3’.
Forward and reverse primer P2F/R is：
Forward primer P2F：5’-CTAGCTAGCTGGCTTCGTCTGAGGCTAT-3’
Reverse primer P2R：5’-CCCAAGCTTCGAACCGCCTTCTCCAAA-3’.
Forward and reverse primer P3F/R is：
Forward primer P3F：5’-CTAGCTAGCTCCACGCCCGGCTCCTCCTC-3’
Reverse primer P3R：5’-CCCAAGCTTCGAACCGCCTTCTCCAAA-3’.
PCR reaction systems are in step (1)：Template DNA 50ng, μ L of 10 × Buffer 2.5, dNTPs concentration are 200 μ
Mo1/L, every primer concentration are 0.4 μm of ol/L, 1U Taq archaeal dna polymerases, add deionized water to the μ l of cumulative volume 25；
PCR reaction conditions are：94 DEG C of pre-degeneration 4min；94 DEG C of denaturation 50s, 60 DEG C of annealing 50s, 72 DEG C of extension 1min, altogether
35 circulations；Last 72 DEG C of extensions 10min；
PCR reaction systems are in step (2)：Template DNA 50ng, μ L of 10 × Buffer 2.5, dNTPs concentration are 200 μ
Mo1/L, every primer concentration are 0.4 μm of ol/L, 1U Taq archaeal dna polymerases, add deionized water to the μ l of cumulative volume 25；
PCR reaction conditions are：94 DEG C of pre-degeneration 4min；94 DEG C of denaturation 50s, 58 or 60 DEG C of annealing 50s, 72 DEG C extend
1min, totally 35 circulations；Last 72 DEG C of extensions 10min.
The technical solution adopted in the present invention also resides in the transfection carrier for providing a kind of promoter of the pig AEBP1 containing regulation and control,
The carrier of the transfection is pGL3-basic.
The technical solution adopted in the present invention, which also resides in, provides a kind of transfection carrier of the promoter of the pig AEBP1 containing regulation and control
Construction method, comprise the following steps：
(1) deletion fragment of pig AEBP1 promoters is connected with pMD-18T carriers, connection product conversion bacillus coli DH 5
α, it is coated on LB flat boards and cultivates, selects positive monoclonal and carry out sequencing identification, the correct positive colony of sequencing result carries out matter
Grain extracting, is named as recombinant plasmid TA-Q, carries out Nhe I and the double digestions of Hind III；
(2) Nhe I and the double digestions of Hind III are carried out to carrier pGL3-basic；
(3) digestion products of step (1) with step (2) are connected, connection product conversion bacillus coli DH 5 alpha, be coated on
Amp+To be cultivated on/LB flat boards, select positive monoclonal and carry out sequencing identification, the correct positive colony of sequencing result expands culture,
And plasmid extraction is carried out, it is named as deleted carrier pGL3-basic-Q.
The reaction system of double digestion is in step (1) and (2)：10×Buffer 2.5-5μL、NheI 0.5-1μL、Hind
III 0.5-1 μ L, the μ L of plasmid vector 5, add ddH2O to 25 μ L；Reaction condition is：37 DEG C of reaction 2h.
The reaction system of connection is in step (3)：10×T4 DNA Ligase Buffer 1μL、T4 DNA Ligase 1
μ L, μ L of pGL3-basic digestion products 0.5, pig AEBP1 gene promoter deletions fragment 7.5 μ L, the μ L of cumulative volume 10；Reaction condition
For：16 DEG C of connection 12h.
The technical solution adopted in the present invention, which also resides in, provides a kind of regulation and control pig AEBP1 promoter in regulation and control pig AEBP1
Application in expression.
The technical solution adopted in the present invention, which also resides in, provides a kind of regulation and control pig AEBP1 promoter in swine improvement
Application.
Beneficial effect of the present invention
(1) present invention is studied pig AEBP1 promoter first, by the different pieces for lacking the gene promoter
Section, obtaining 3 kinds has more strongly active new promoter.The new promoter of the present invention can regulate and control the table of pig AEBP1 genes
Reach, new instrument and selection are provided for swine improvement.
(2) biological experiment shows, the present invention build 3 deleted carrier pGL3-basic-Q1, pGL3-basic-Q2,
PGL3-basic-Q3 has more strongly active, wherein pGL3-basic-Q1 active highest in Ren sus domestica cell PK-15.This hair
Bright result finally obtains 1483bp promoter fragment (- 337bp arrives -1819bp), i.e., the recombination expression that the present invention is built carries
The promoter fragment included in body pGL3-basic-Q1, possesses independent startup function.
Brief description of the drawings
The embodiment of the present invention is described in further detail below in conjunction with accompanying drawing.
Fig. 1 is Large White DNA electrophoresis detection results, in figure,
1 swimming lane is Large White DNA, and 2 swimming lanes are DL2000 standard molecular weights.
Fig. 2 is carrier pGL3-basic structural representation.
Fig. 3 is carrier pGL3-basic-Q1, pGL3-basic-Q2, pGL3-basic-Q3 double digestion qualification result, figure
In,
A, b, c are respectively carrier pGL3-basic-Q1, pGL3-basic-Q2, pGL3-basic-Q3 double digestion identification knot
Fruit, 1 swimming lane are the double digestion product of corresponding carrier, and 2 swimming lanes are DL2000 standard molecular weights.
Fig. 4 is Dual-Luciferase Activity determination result.
Embodiment
The embodiment of the present invention is described in further detail with reference to embodiments.
The acquisition of the corresponding deletion fragment of the pig AEBP1 gene promoters of embodiment 1
1st, Large White DNA extraction
1.1st, DNA is extracted
Large White (kind routinely reported) new blood is gathered, 0.5M ethylenediamine tetra-acetic acids (EDTA) is added and is used as anti-freezing
Agent (blood flow volume：EDTA volume ratios are 5：1), shake up.
Leucocyte separates：
(1) 10min is centrifuged in 4 DEG C of 6000rpm, abandons upper serum.
(2) 1.5 times of volume distilled waters are added, gently shake up 10min.
(3) 4 DEG C of 5000rpm centrifuge 10min, go to upper strata.
(4) mass fraction 0.9%NaCl solution washing precipitation is added, gently shakes up 10min.
(5) 4 DEG C of 5000rpm centrifuge 10min, remove supernatant, and it is leucocyte to obtain precipitation.
1.2nd, Genome DNA extraction：
(1) in leukocyte cell pellet, isometric 1 × SET buffer solutions, Proteinase K (10mg/mL) to the μ of final concentration 100 are added
G/mL, the lauryl sodium sulfate of mass concentration 10% (SDS) to final concentration 0.5%, shakes up, and is incubated overnight in 55 DEG C of shaking baths
Digestion.
(2) isometric Tris saturated phenols are added, gently shake up 15min, 10min is centrifuged in 4 DEG C of 11000rpm, draws supernatant
Liquid is transferred to new centrifuge tube.
(3) isometric phenol/chloroform/isoamyl alcohol mixed liquor (volume ratio 25 is added：24：1) centrifuge tube, is jiggled
10min, 4 DEG C of 11000rpm centrifuge 10min, supernatant are drawn to new centrifuge tube.
(4) isometric chloroform/isoamyl alcohol (volume ratio 24 is added：1) centrifuge tube 10min, 4 DEG C of 11000rpm, are jiggled
Centrifuge 10min, Aspirate supernatant to new centrifuge tube.
(5) absolute ethyl alcohol of 2.5 times of volume precoolings is added, gently shakes centrifuge tube, visible white is cotton-shaped after careful mixing
DNA is precipitated.
(6) suction out cotton-shaped DNA in the centrifuge tube of a 1.5ml with suction nozzle, washed once with 70% ethanol, 3000rpm from
Heart 5min, abandon upper strata ethanol, dry DNA.
(7) determine DNA concentration and purity, and by the agarose gel electrophoresis of concentration 1%, DNA bands are detected under uviol lamp
Size (see Fig. 1).
2nd, the acquisition of pig AEBP1 gene promoters
2.1st, primer is designed
Utilize DNA sequence dna (the GenBank accession number of the pig AEBP1 genes on NCBI：DQ464432 it is) probe sequence,
NCBI(http://www.ncbi.nlm.nih.gov/) compare, obtain purpose fragment (GenBank accession number：CU928907),
PCR primer is designed according to purpose fragment, enters performing PCR and expands this section of sequence, the primer is as follows：
Forward primer F：5’-GTGGCTGCCTGTTCATCATCG-3’(SEQ ID NO.1)
Reverse primer R：5’-CTTTGCGGACTGGAAGTGTG-3’(SEQ ID NO.2)
2.2nd, PCR is expanded
PCR reaction systems are：Template DNA 50ng, μ L of 10 × Buffer 2.5, dNTPs concentration are 200 μm of o1/L, every
0.4 μm of ol/L of primer concentration, 1U Taq archaeal dna polymerases, add deionized water to the μ l of cumulative volume 25.
PCR reaction conditions are：94 DEG C of pre-degeneration 4min；94 DEG C of denaturation 50s, 60 DEG C of annealing 50s, 72 DEG C of extension 1min, altogether
35 circulations；Last 72 DEG C of extensions 10min.
3rd, the acquisition of pig AEBP1 gene promoter deletions fragment
3.1st, primer is designed
PCR primer is sequenced, selects First Exon ATG upstream 2000bp sequences as promoter candidate segment,
It is as follows：
GATTTTCCCACCTGGGCCCAACCCTCACAGACCACGTTGGCTCAGGACTGACCGCTGCCAGACTTGGCCCGTGGACT
CCAGATGTGCTTTTTCTTGCTTCCTCATTCCTTCAAGGAGGAATGTTCTGGGAACTCTGGCCTTAGGTGTCATGCCT
ATTTGGAACCAGCCAGCATTTGCCCTTCGAGAAGCCTTATGGAAACACCAGGCACACGTGGCATAGCTCCCTATGCC
AGCACCCATGGCAGACATCACTAATCGATCCTGGGATTCTCTGACTACCCACTCTCCCTGTGCCCAGGGCCCATGTG
GTAGCTGGAGTTATTGTAAAGACAATTTTTAAAAACTGGGTTCTGGTCAGAGGAAGGAGAGGAATGGCTCTTTGCCC
TTGTTGGGGTCTCAGCTGGTGGGAAAGCAGTAGCCTTAGAGGTGAAGCCATGTTCATCACGGGAAATAGTAAAGGAA
ACTCTAGGGGAGGGGACCTGACCCCCCCACATCTGTGGCTCCTGTTTGGGTGGGGAGTAAACCGTTCCCAGAGCAGA
TCCCGGCACACAGCTTCCTTGGGTTGAAGGCACCCATCCTCTCTCCCCAGCCGATCCATGTCCTCAGGGGTGCTGTG
TGACCCCCTATGCCTTTGTGGCCCCTCCATCTTCCACACAGAGCTGGGCACACACACACTGCCCCTAAGATATTCCC
ATGACTTGACCTGGCTTCGTCTGAGGCTATGTCACCAGCTAGAGACAGGCCAGGTAAGTGGGCTGCTGCCCACCATC
TCCTTGCTGGGGATCTTACCTGGGATCCCCTCTCCTGCTGCTGGGAGCACAGGGGTGGGCCAGAAGCCCCCGAACTT
GTACGGAAATCGCTGCGTGAATGCCGAGTCCCTCCCCACTTGTGACAGTCCATTCCTATGAATGACACATACCAGTT
GATAAAACATGAAATCCCCTCCCTCCCTGCTCCAACGCTGACCCCGCTCCAAACCTGGCCTTGTGCATCAGGACCTT
GCGGGTGTGGGGGTCTCCTGCCCTGAGTGAGACCAGCTGTGCACCTGCCCCTTACTAGACAGTGAATTCCCAAAATG
TGGTCCCTCCCTGGGGCCAACACAGGGCCCCGGGCTCTTCATGGACCTCAGAGGTTAGCTGGTTGCCTGAAGGATGG
CTGGTGACTGCCGAGCTGCCAGGGCAAGGCTCCAGAGAGAGGAAAGCATGGGTTGGCCCTCAAGGTCCTTCTGAGAT
GGGGATGGTCCCCAAGGCCACCGTCCCTGGCTTGGCTCTGCTCTCAGGGTTCCCTGGTCGCCTCCAGGTTGCCGTCG
GGCTTGGGAGCGTGCCACAGAGTGAGTGAGACAGGGGCGTGGGGGCGCTGAGACCCACGCTTTGGGGTCCCCCTGCA
TCCTCTCCCACCCTCCCTCCTACAGGCCTTCCTTCCCAGGCGACCACCCCCCCGCCCCCGCCCCCGCCAGCCCGGGG
ACACCGCCCGCAGTTCCCACTTCGGGAAATGCCAAGCGCGCGGCGCCGCCGGGACAGCTCAGCAGCCCCGCAGCCCC
TCCGTCCACGCCCGGCTCCTCCTCCTCCTCCTCCTCCTCCTTCCCCGGGGGTGGGGGGAGCCGGCTCCGGAAGCCCC
GGCGCCCCCGCCCGGGCCGCCACTGGCGCTTTGGAGAAGGCGGTTCGCGTGCCACGCGGGACCTGAGCTCCCAGCCG
CCGCCAGCTCCTTAGGAACCCCCTGACCCCTTCGGAACCCCCACCGACTCCCGAGCCACTGCCCGGCCCGGAGCTAG
CTCCCCGCGCCCCACCCCGGAGTCCCCTGCTACCCCAGATCTCCCCCTCCGCCCCCACCCCTCCCAAGAGCCCACCC
CGATCCCCTCCGCCCGCCCCTCCCCGGACCTCCCCACCCACCCCCGATCCTCCCACCCACCCCTCCCAGGAACCCCT
ACCCCCGCTCCCAGGACCCCGCGGCACCCCTTCCCCGGAGCCCCCCGCGCTCCTGGCCCGGTGCGCGCCGCGGCC
(SEQ ID NO.3)
Design 3 deletion fragment (P1, P2, P3) amplimer P1F/R, P2F/R, P3F/R (SEQ ID NO.4-SEQ ID
NO.9), 35 of structure are represented successively’The forward primer and reverse primer of deleted carrier are held, wherein, added before forward primer
The restriction enzyme sites of Nhe I (GCTAGC, being represented with underscore), while add protectiveness base CTA (shadow representation)；Reverse primer adds
Add the restriction enzyme sites of Hind III (AAGCTT, being represented with underscore), while add protectiveness base CCC (shadow representation), primer sequence
Row are shown in Table 1.
The design of the promoter deletion vector primer of table 1
3.2nd, PCR is expanded
Enter performing PCR amplification to promoter candidate segment.
PCR reaction systems are：Template DNA 50ng, μ L of 10 × Buffer 2.5, dNTPs concentration are 200 μm of o1/L, every
0.4 μm of ol/L of primer concentration, 1U Taq archaeal dna polymerases, add deionized water to the μ l of cumulative volume 25.
PCR reaction conditions are：94 DEG C of pre-degeneration 4min；94 DEG C of denaturation 50s, 58 or 60 DEG C of annealing 50s (as shown in table 1),
72 DEG C of extension 1min, totally 35 circulations；Last 72 DEG C of extensions 10min.PCR corresponding to amplimer P1F/R, P2F/R, P3F/R
Amplified fragments are as follows.
P1F/R：
CGAGAAGCCTTATGGAAACACCAGGCACACGTGGCATAGCTCCCTATGCCAGCACCCATGGCAGACATCACTAATCG
ATCCTGGGATTCTCTGACTACCCACTCTCCCTGTGCCCAGGGCCCATGTGGTAGCTGGAGTTATTGTAAAGACAATT
TTTAAAAACTGGGTTCTGGTCAGAGGAAGGAGAGGAATGGCTCTTTGCCCTTGTTGGGGTCTCAGCTGGTGGGAAAG
CAGTAGCCTTAGAGGTGAAGCCATGTTCATCACGGGAAATAGTAAAGGAAACTCTAGGGGAGGGGACCTGACCCCCC
CACATCTGTGGCTCCTGTTTGGGTGGGGAGTAAACCGTTCCCAGAGCAGATCCCGGCACACAGCTTCCTTGGGTTGA
AGGCACCCATCCTCTCTCCCCAGCCGATCCATGTCCTCAGGGGTGCTGTGTGACCCCCTATGCCTTTGTGGCCCCTC
CATCTTCCACACAGAGCTGGGCACACACACACTGCCCCTAAGATATTCCCATGACTTGACCTGGCTTCGTCTGAGGC
TATGTCACCAGCTAGAGACAGGCCAGGTAAGTGGGCTGCTGCCCACCATCTCCTTGCTGGGGATCTTACCTGGGATC
CCCTCTCCTGCTGCTGGGAGCACAGGGGTGGGCCAGAAGCCCCCGAACTTGTACGGAAATCGCTGCGTGAATGCCGA
GTCCCTCCCCACTTGTGACAGTCCATTCCTATGAATGACACATACCAGTTGATAAAACATGAAATCCCCTCCCTCCC
TGCTCCAACGCTGACCCCGCTCCAAACCTGGCCTTGTGCATCAGGACCTTGCGGGTGTGGGGGTCTCCTGCCCTGAG
TGAGACCAGCTGTGCACCTGCCCCTTACTAGACAGTGAATTCCCAAAATGTGGTCCCTCCCTGGGGCCAACACAGGG
CCCCGGGCTCTTCATGGACCTCAGAGGTTAGCTGGTTGCCTGAAGGATGGCTGGTGACTGCCGAGCTGCCAGGGCAA
GGCTCCAGAGAGAGGAAAGCATGGGTTGGCCCTCAAGGTCCTTCTGAGATGGGGATGGTCCCCAAGGCCACCGTCCC
TGGCTTGGCTCTGCTCTCAGGGTTCCCTGGTCGCCTCCAGGTTGCCGTCGGGCTTGGGAGCGTGCCACAGAGTGAGT
GAGACAGGGGCGTGGGGGCGCTGAGACCCACGCTTTGGGGTCCCCCTGCATCCTCTCCCACCCTCCCTCCTACAGGC
CTTCCTTCCCAGGCGACCACCCCCCCGCCCCCGCCCCCGCCAGCCCGGGGACACCGCCCGCAGTTCCCACTTCGGGA
AATGCCAAGCGCGCGGCGCCGCCGGGACAGCTCAGCAGCCCCGCAGCCCCTCCGTCCACGCCCGGCTCCTCCTCCTC
CTCCTCCTCCTCCTTCCCCGGGGGTGGGGGGAGCCGGCTCCGGAAGCCCCGGCGCCCCCGCCCGGGCCGCCACTGGC
GCTTTGGAGAAGGCGGTTCG(SEQ ID NO.10)
P2F/R：
TGGCTTCGTCTGAGGCTATGTCACCAGCTAGAGACAGGCCAGGTAAGTGGGCTGCTGCCCACCATCTCCTTGCTGGG
GATCTTACCTGGGATCCCCTCTCCTGCTGCTGGGAGCACAGGGGTGGGCCAGAAGCCCCCGAACTTGTACGGAAATC
GCTGCGTGAATGCCGAGTCCCTCCCCACTTGTGACAGTCCATTCCTATGAATGACACATACCAGTTGATAAAACATG
AAATCCCCTCCCTCCCTGCTCCAACGCTGACCCCGCTCCAAACCTGGCCTTGTGCATCAGGACCTTGCGGGTGTGGG
GGTCTCCTGCCCTGAGTGAGACCAGCTGTGCACCTGCCCCTTACTAGACAGTGAATTCCCAAAATGTGGTCCCTCCC
TGGGGCCAACACAGGGCCCCGGGCTCTTCATGGACCTCAGAGGTTAGCTGGTTGCCTGAAGGATGGCTGGTGACTGC
CGAGCTGCCAGGGCAAGGCTCCAGAGAGAGGAAAGCATGGGTTGGCCCTCAAGGTCCTTCTGAGATGGGGATGGTCC
CCAAGGCCACCGTCCCTGGCTTGGCTCTGCTCTCAGGGTTCCCTGGTCGCCTCCAGGTTGCCGTCGGGCTTGGGAGC
GTGCCACAGAGTGAGTGAGACAGGGGCGTGGGGGCGCTGAGACCCACGCTTTGGGGTCCCCCTGCATCCTCTCCCAC
CCTCCCTCCTACAGGCCTTCCTTCCCAGGCGACCACCCCCCCGCCCCCGCCCCCGCCAGCCCGGGGACACCGCCCGC
AGTTCCCACTTCGGGAAATGCCAAGCGCGCGGCGCCGCCGGGACAGCTCAGCAGCCCCGCAGCCCCTCCGTCCACGC
CCGGCTCCTCCTCCTCCTCCTCCTCCTCCTTCCCCGGGGGTGGGGGGAGCCGGCTCCGGAAGCCCCGGCGCCCCCGC
CCGGGCCGCCACTGGCGCTTTGGAGAAGGCGGTTCG(SEQ ID NO.11)
P3F/R：
TCCACGCCCGGCTCCTCCTCCTCCTCCTCCTCCTCCTTCCCCGGGGGTGGGGGGAGCCGGCTCCGGAAGCCCCGGCG
CCCCCGCCCGGGCCGCCACTGGCGCTTTGGAGAAGGCGGTTCG(SEQ ID NO.12)
The transfection carrier structure of the corresponding deletion fragment of promoter of the pig AEBP1 genes of embodiment 2
1st, recombinant plasmid TA-Q structure
3 pig AEBP1 gene delections fragment PCR products recovery that embodiment 1 is obtained is (with reference to the recovery of Shanghai Lai Feng companies
Kit specification), it is respectively connecting to pMD-18T carriers (with reference to TAKARA company pMD-18T carriers operation instructions), connection
Product converts bacillus coli DH 5 alpha, and step is as follows：
(1) 5 μ L connection product is drawn in 100 μ L competent cells, is mixed, is placed 30min on ice.
(2) 42 DEG C of water-baths, thermal shock 90s are inserted.
(3) pipe is taken out rapidly and be placed on ice cube, cool down 3-4min.
(4) LB fluid nutrient mediums of the 400 μ L without ampicillin (Amp) is added in pipe, 37 DEG C of recovery 1h.
(5) 5min is centrifuged in 4500rpm, part supernatant is suctioned out, remaining part mixes, and is transferred to LB culture medium flat plates
Upper (containing IPTG and X-gal), pave, after liquid is absorbed, 37 DEG C are inverted culture 12-16h.
Select positive monoclonal and carry out sequencing identification.The correct positive colony of sequencing result, plasmid extraction is carried out, is named as
Recombinant plasmid TA-Q1, TA-Q2, TA-Q3.
2nd, deleted carrier pGL3-basic-Q structure
By recombinant plasmid TA-Q1, TA-Q2, TA-Q3 respectively with III pair of enzyme of corresponding restriction enzyme Nhe I and Hind
Cut.Carrier pGL3-basic (being purchased from Pu Luomaige Beijing Bioisystech Co., Ltd) also carries out the Hes of restriction enzyme Nhe I
The double digestions of Hind III, carrier pGL3-basic structure are shown in Fig. 2.Digestion system is as shown in table 2：
The endonuclease reaction system of table 2
| 10×Buffer | 2.5-5μL |
| NheI | 0.5-1μL |
| HindⅢ | 0.5-1μL |
| Plasmid vector | 5μL |
| Add ddH2O | To 25 μ L |
After 37 DEG C of digestion 2h, recovery purpose fragment (is returned with reference to the glue of Shanghai Lay maple biological gene technology Co., Ltd
Receive kit specification operation), it is placed in -20 DEG C of refrigerators and preserves.
The pig AEBP1 gene promoter deletion fragments that digestion is reclaimed are connected on pGL3-basic carrier digestion products.
Linked system is as shown in table 3：
The coupled reaction system of table 3
| 10×T4 DNA Ligase Buffer | 1μL |
| T4 DNA Ligase | 1μL |
| PGL3-basic digestion products | 0.5μL |
| Pig AEBP1 gene promoter deletion fragments | 7.5μL |
| Cumulative volume | 10μL |
Coupled reaction condition is 16 DEG C of water-bath 12h.Connection product is converted into bacillus coli DH 5 alpha, is coated on Amp+/ LB is put down
(the LB flat boards i.e. containing Amp) is cultivated on plate, is selected positive monoclonal and is carried out sequencing identification.Correct positive colony will be sequenced to expand
Culture, plasmid extraction (by the small amount plasmid extraction agent box D6950-01 specifications operation of OMEGA companies) is carried out, is respectively designated as
Deleted carrier pGL3-basic-Q1, pGL3-basic-Q2, pGL3-basic-Q3.With Nhe I and the double digestions of Hind III identification matter
Grain (result is shown in Fig. 3), digestion system is the same as table 2.Determine concentration (the Beckman companies of the U.S. of plasmidNucleic acid-protein
Concentration mensuration instrument), it is standby to be placed in -20 DEG C of refrigerators.
The liposome-mediated cell transfecting of embodiment 3
Liposome-mediated cell transfecting specific steps are with reference to LipofectamineTM2000 (invitrogen companies)
Specification, transfection are carried out with 24 hole Tissue Culture Dish, and the cell used is porcine kidney cell PK-15.To eliminate experimental error, each
Deleted carrier (pGL3-basic-Q1, pGL3-basic-Q2, pGL3-basic-Q3) carries out three-wheel independent experiment, every time experiment
Three multiple holes.1 μ g deleted carriers, 3 μ L liposomes are used per hole.After collecting cell pyrolysis liquid after transfectional cell 48h, by double glimmering
Light element enzyme detecting system carries out the activity analysis of deletion promoters.
Being formulated as follows for the step of being related to porcine kidney cell PK-15 culture, transfection in the present invention and related reagent is described：
(1) preparation of related reagent
A) cell culture fluid (hyclone concentration is 10% culture medium, by taking 20mL volumes as an example)：Hyclone FBS
(being purchased from GIBCO companies) melts in 4 DEG C of refrigerators, and hyclone 2mL, penicillin concn 100U/mL, streptomysin concentration are 100 μ
G/mL, DMEM/F12 culture medium (being purchased from invitrogen companies) are settled to 20mL, standby in 4 DEG C.
B) cell dissociation buffer：Be purchased from GIBCO companies, be placed in after sterilization 4 DEG C it is standby.
C) balanced salt solution PBS：Be purchased from GIBCO companies, be placed in after sterilization 4 DEG C it is standby.
(2) cell culture (porcine kidney cell PK-15)
A) change cell culture fluid within every two days, should be changed in time when turning yellow such as cell culture fluid.
B) when cell confluency degree is up to passing on after 80%, old culture medium is discarded, with 1 × PBS (being purchased from invitrogen companies)
Wash twice.
C) the 0.25% trypsin solution 1mL for preheating 37 DEG C is added into 50cm2Tissue Culture Flask (is purchased from invitrogen
Company), ensure that trypsase covers all cells, be placed in about 1-2min in 37 DEG C of incubators, observe cell state in time.
D) cell loosen and from culture dish show to come off when, add 10%FBS cell growth medium (being purchased from GIBCO companies)
Terminate the effect of trypsase.
E) piping and druming allows cell fully to come off and forms uniform cell suspension repeatedly.
F) according to 1:3 are passed on, and it is 4mL to add cell growth medium to final volume.
(3) cell transfecting
A) pass on：In the day before transfection, take the good cell of one bottle of upgrowth situation, added after Trypsin Induced 2mL without
Antibiotic cell culture fluid (10% FBS), dispel as uniform cell suspension, take 10 μ L to be counted by cell counting count board
Number.Appropriate cell is taken, supplement is without antibiotic cell growth nutrient solution (10% FBS) to 12mL, the packing 500 per hole after mixing
μ L, 24 porocyte culture plates are about 0.5-2 × 10 per hole cell number5, shake up and be placed in 37 DEG C, 5%CO2Trained in cell culture incubator
Support 24h.
B) transfect：1 μ g deleted carriers and 50 μ L OPTI-MEM nutrient solutions are taken (invitrogen companies to be purchased from, by the reagent
Specification operation) mix after stand 5min；3 μ L LipofectamineTM2000 and 50 μ L OPTI-MEM nutrient solutions (carry
Weight：LipofectamineTM2000 volumes are 1 μ g:3 μ L), it is stored at room temperature 5min after well mixed.
C) in 30min, two mixed liquors in step b) is well mixed, are stored at room temperature 20min.
D) cell original fluid in step a) is abandoned, is discarded afterwards twice with 1 × PBS cell.
E) step c) the μ L of mixed liquor 100 are added into every hole cell, OPTI-MEM nutrient solutions is reused and mends to cumulative volume
For 500 μ L, shake up.
F) cell culture incubator culture (37 DEG C, 5%CO2) 6h, it is replaced with the cell growth nutrient solution (10% without antibiotic
FBS)。
Embodiment 4 pig AEBP1 gene promoters, 3 deletion fragment Dual-Luciferase Activity determinations
3 deletion fragment Dual-Luciferase activity of pig AEBP1 gene promoters are detected, negative control vector is set
PGL3-basic, positive control plasmid pGL3-control, internal reference plasmid (to correct transfection efficiency) use sea pansy fluorescein
Enzyme reporter gene PRL-TK [Pu Luomaige (Beijing) Bioisystech Co., Ltd, i.e. U.S. Promega companies], transfection reagent makes
Use LipofectamineTM2000.24 hole Tissue Culture Dish are pressed per hole carrier quality：The μ g of liposome volume ratio 1：3 μ L, with sky
Transfection (carrier pGL3-basic) is compareed, and 3 deleted carriers of structure are transiently transfected into porcine kidney cell PK-15 respectively, treat 48h
After collect cell pyrolysis liquid, by luciferase reporter gene detecting system to 3 deletion fragments of pig AEBP1 gene promoters
Activity analysis is carried out, to determine high activity section (result is shown in Fig. 4).As a result show：Compared with negative control pGL3-basic, structure
3 deleted carriers pGL3-basic-Q1, pGL3-basic-Q2, the pGL3-basic-Q3 built have stronger in PK-15 cells
The active highest of activity, wherein pGL3-basic-Q1.The result of the present invention finally obtains 1483bp promoter fragment (i.e. originally
Invent deleted carrier pGL3-basic-Q1, the SEQ ID NO.10 of structure) possess independent startup function.
Claims (10)
1. a kind of regulation and control pig AEBP1 promoter, it is characterised in that the promoter is SEQ ID NO.10, SEQ ID
Nucleotide sequence shown in NO.11 or SEQ ID NO.12.
2. a kind of construction method of regulation and control pig AEBP1 as claimed in claim 1 promoter, it is characterised in that including following
Step：
(1) with GenBank accession number：CU928907 nucleotides sequence is classified as template, designs forward and reverse primers F/R, and PCR is expanded,
Amplified fragments First Exon ATG upstreams 2000bp sequences are selected as promoter candidate segment；
(2) using promoter candidate segment as template, design 3 deletion fragments P1, P2, P3 forward and reverse primer P1F/R, P2F/R,
P3F/R, enter performing PCR amplification respectively, obtain pcr amplification product, as regulate and control pig AEBP1 promoter.
3. the construction method of regulation and control pig AEBP1 according to claim 2 promoter, it is characterised in that
Forward and reverse primers F/R is：
Forward primer F：5’-GTGGCTGCCTGTTCATCATCG-3’
Reverse primer R：5’-CTTTGCGGACTGGAAGTGTG-3’
Forward and reverse primer P1F/R is：
Forward primer P1F：5’-CTAGCTAGCCGAGAAGCCTTATGGAAACA-3’
Reverse primer P1R：5’-CCCAAGCTTCGAACCGCCTTCTCCAAA-3’
Forward and reverse primer P2F/R is：
Forward primer P2F：5’-CTAGCTAGCTGGCTTCGTCTGAGGCTAT-3’
Reverse primer P2R：5’-CCCAAGCTTCGAACCGCCTTCTCCAAA-3’
Forward and reverse primer P3F/R is：
Forward primer P3F：5’-CTAGCTAGCTCCACGCCCGGCTCCTCCTC-3’
Reverse primer P3R：5’-CCCAAGCTTCGAACCGCCTTCTCCAAA-3’.
4. the construction method of regulation and control pig AEBP1 according to claim 2 promoter, it is characterised in that
PCR reaction systems are in step (1)：Template DNA 50ng, μ L of 10 × Buffer 2.5, dNTPs concentration are 200 μm of o1/
L, every primer concentration is 0.4 μm of ol/L, 1U Taq archaeal dna polymerases, adds deionized water to the μ l of cumulative volume 25；
PCR reaction conditions are：94 DEG C of pre-degeneration 4min；94 DEG C denaturation 50s, 60 DEG C annealing 50s, 72 DEG C extension 1min, totally 35
Circulation；Last 72 DEG C of extensions 10min；
PCR reaction systems are in step (2)：Template DNA 50ng, μ L of 10 × Buffer 2.5, dNTPs concentration are 200 μm of o1/
L, every primer concentration is 0.4 μm of ol/L, 1U Taq archaeal dna polymerases, adds deionized water to the μ l of cumulative volume 25；
PCR reaction conditions are：94 DEG C of pre-degeneration 4min；94 DEG C of denaturation 50s, 58 or 60 DEG C of annealing 50s, 72 DEG C of extension 1min, altogether
35 circulations；Last 72 DEG C of extensions 10min.
A kind of 5. transfection carrier of the promoter of the regulation and control pig AEBP1 containing described in claim 1, it is characterised in that the transfection
Carrier be pGL3-basic.
6. a kind of construction method of the transfection carrier of regulation and control pig AEBP1 as claimed in claim 5 promoter, its feature exist
In comprising the following steps：
(1) deletion fragment of pig AEBP1 promoters is connected with pMD-18T carriers, connection product conversion bacillus coli DH 5 alpha, applied
Cloth is cultivated on LB flat boards, is selected positive monoclonal and is carried out sequencing identification, the correct positive colony of sequencing result carries out plasmid and taken out
Carry, be named as recombinant plasmid TA-Q, carry out Nhe I and the double digestions of Hind III；
(2) Nhe I and the double digestions of Hind III are carried out to carrier pGL3-basic；
(3) digestion products of step (1) with step (2) are connected, connection product conversion bacillus coli DH 5 alpha, is coated on Amp+/LB
Cultivated on flat board, select positive monoclonal and carry out sequencing identification, the correct positive colony of sequencing result expands culture, and carries out matter
Grain extracting, is named as deleted carrier pGL3-basic-Q.
7. the construction method of the transfection carrier of regulation and control pig AEBP1 according to claim 6 promoter, it is characterised in that
The reaction system of double digestion is in step (1) and (2)：10×Buffer 2.5-5μL、NheI 0.5-1μL、HindⅢ0.5-1μ
L, the μ L of plasmid vector 5, add ddH2O to 25 μ L；Reaction condition is：37 DEG C of reaction 2h.
8. the construction method of the transfection carrier of regulation and control pig AEBP1 according to claim 6 promoter, it is characterised in that
The reaction system of connection is in step (3)：10×T4DNA Ligase Buffer 1μL、T4DNA Ligase 1μL、pGL3-
μ L of basic digestion products 0.5, pig AEBP1 gene promoter deletions fragment 7.5 μ L, the μ L of cumulative volume 10；Reaction condition is：16℃
Connect 12h.
A kind of 9. application of regulation and control pig AEBP1 as claimed in claim 1 promoter in regulation and control pig AEBP1 expression.
A kind of 10. application of regulation and control pig AEBP1 as claimed in claim 1 promoter in swine improvement.
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| Sus scrofa chromosome 18 clone CH242-155A21, WORKING DRAFT SEQUENCE, 2 unordered pieces;McLaren,S.;《GenBank: CU928907》;20090110;全文 * |
| 猪脂肪细胞分化、脂肪沉积相关候选基因的分离、定位及遗传效应分析;陈俊峰;《中国优秀博硕士学位论文全文数据库（博士）农业科技辑》;20070215;第99-102页小结与下一步工作部分 * |
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