CN108384790A - The method of the identification regulation and control active controlling element of FATP1 gene promoter transcriptions - Google Patents
The method of the identification regulation and control active controlling element of FATP1 gene promoter transcriptions Download PDFInfo
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Abstract
The method that the present invention provides the identification regulation and control active controlling element of FATP1 gene promoter transcriptions, includes the following steps：Clone FATP1 gene promoter sequences；It builds series of deletions FATP1 genes 5 ' and holds promoter fragment luciferase reporter vector；Above-mentioned luciferase reporter vector is distinguished into transfectional cell, uciferase activity analysis is carried out, determines FATP1 gene core promoters active region；Binding site for transcription factor in the active region is analyzed, rite-directed mutagenesis is carried out to Binding site for transcription factor, according to FATP1 gene promoter activities situation of change after mutation, determines regulation and control FATP1 gene promoter transcription activity regulation elements.The present invention identifies regulation and control FATP1 gene promoter transcription activity regulation elements, is finally determined that KLF15 Binding site for transcription factor is the regulation and control active controlling element of FATP1 gene promoter transcriptions.
Description
Technical field
The present invention relates to the methods of the identification regulation and control active controlling element of FATP1 gene promoter transcriptions.
Background technology
Fatty acid transport protein 1 (Fatty acid transport protein 1, FATP1) is as promotion long-chain fat
The integral membrane proteins of acid intake, participate in oleic acid synthesis regulation, are one of the target genes for improving beef quality.Oleic acid is as main single
Palmitic acid in one unsaturated fatty acid accounts for the 33% of beef lipid, inhibited to serum cholesterol concentration.Insulin
It can induce FATP1 around nucleus and intracellular ectopic expression, long chain fatty acids (LCFA) intake is made to increase.
FATP1 genes high abundance in the high tissue of the fatty acid metabolisms such as heart, skeletal muscle, fat is expressed.FATP1 gene expression amounts
Increase and shows identical trend, and its polymorphism and oleic acid with the increase of oleic acid intake during preadipocyte differentiation
Composition ratio is in significantly correlated.The content of FATP1 gene expression amounts and intramuscular fat in beef skeletal muscle is in notable positive correlation.To the greatest extent
Unsaturated fatty acid forms in pipe FATP1 Gene Expression bovine muscles, but the transcriptional control about ox FATP1 genes at present
Mechanism, which has no, reports for work.
Invention content
In order to solve the problems in the existing technology, the present invention provides a kind of identifications to regulate and control FATP1 gene promoters
The method of the controlling element of transcriptional activity, and it is determined that KLF15 Binding site for transcription factor is that regulation and control FATP1 gene promoters turn
Record active controlling element.
The first purpose of the invention is to provide the sides of the identification regulation and control active controlling element of FATP1 gene promoter transcriptions
Method includes the following steps：
(1) FATP1 gene promoter sequences are cloned；
(2) structure FATP1 genes 5 ' hold promoter series of deletions segment luciferase reporter vector；
(3) by the FATP1 genes 5 ' of step (2) hold promoter series of deletions segment luciferase reporter vector respectively into
Row transfectional cell carries out uciferase activity analysis, determines FATP1 gene core promoters active region；
(4) Binding site for transcription factor in analysis FATP1 gene core promoters active region, combines transcription factor
Site carries out rite-directed mutagenesis, and regulation and control FATP1 gene promoters are determined according to FATP1 gene promoter activities situation of change after mutation
The controlling element of transcriptional activity：After Binding site for transcription factor mutation, starts activity and be remarkably decreased, illustrate the transcription factor knot
It is the regulation and control active controlling element of FATP1 gene promoter transcriptions to close site.
Preferably, clone's FATP1 gene promoter sequences, build FATP1 gene promoter series of deletions segments
Luciferase reporter vector includes the following steps：
(1) according to 5 ' UTR sequence of FATP1 genes, the transcription initiation site identified with 5 '-RACE designs FATP1 for+1
Gene promoter specific primer carries out PCR amplification to FATP1 gene promoters, and target fragment is from promoter sequence -1856
Position arrives+190；
(2) pcr amplification product glue is recycled, and withSimple carriers connect, transformed competence colibacillus cell into
Row culture, is sequenced positive colony bacterium solution, then extracts plasmid to the correct bacterium solution of sequencing result；Design primer and more than
It is template to state the correct bacterium solution of sequencing, and amplification FATP1 genes 5 ' hold promoter series of deletions segment；
(3) promoter series of deletions segment and luciferase reporter vector point are held to the FATP1 genes 5 ' of step (2) amplification
It is connected after other digestion, connection product is converted, positive colony bacterium solution is sequenced, then to the correct bacterium of sequencing result
Liquid extracts plasmid, obtains luciferase reporter vector：pGL-1856/+190(P1)、pGL-1558/+190(P2)、pGL-1261/
+190(P3)、pGL-955/+190(P4)、pGL-640/+190(P5)、pGL-387/+190(P6)、pGL-96/+190(P7)。
Preferably, specific primer described in step (1) is：
Sense primer is F1:5′-CTACTGTGGTGGGCACTTG-3′；
Downstream primer is R1：5′-TTGTTCCCTGGCTGACCTGGAG-3′.
Preferably, FATP1 genes 5 ' described in step (2) hold promoter series of deletions segment luciferase reporter vector
For：pGL-1856/+190(P1)、pGL-1558/+190(P2)、pGL-1261/+190(P3)、pGL-955/+190(P4)、
pGL-640/+190(P5)、pGL-387/+190(P6)、pGL-96/+190(P7)。
Preferably, the luciferase reporting of the end of FATP1 genes 5 ' described in step (3) promoter series of deletions segment carries
Body transfectional cell is transfected C2C12 cell lines and 3T3-L1 cell lines.
Preferably, the luciferase reporting of the end of FATP1 genes 5 ' described in step (3) promoter series of deletions segment carries
Use pRL-TK as internal reference plasmid while body transfectional cell, pGL3-Basic is as negative control plasmids, pGL3-Control
As positive control plasmid.
Second object of the present invention is to provide KLF15 Binding site for transcription factor and is maintaining FATP1 gene promoter subbases
Application in this transcriptional activity.
The present invention is by uciferase activity analysis method to regulating and controlling the active controlling element of FATP1 gene promoter transcriptions
It is identified, is finally determined that KLF15 Binding site for transcription factor is the regulation and control active regulation and control of FATP1 gene promoter transcriptions
Element.
Description of the drawings
Attached drawing is used to provide further understanding of the present invention, and a part for constitution instruction, the reality with the present invention
It applies example to be used to explain the present invention together, not be construed as limiting the invention.In the accompanying drawings：
Fig. 1 is FATP1 gene promoter amplifications；Wherein, M:DL2000DNA Marker；1, FATP1 gene promoter
Sub- PCR product.
Fig. 2 identifies for pGL-FATP1 different fragments Sac I and Xho I double digestions；Wherein, M:DL2000DNA Marker；
P1：pGL-1856/+190；P2：pGL-1558/+190；P3：pGL-1261/+190；P4：pGL-955/+190；P5：pGL-
640/+190；P6：pGL-387/+190；P7：pGL-96/+190.
Fig. 3 is that gene promoter recombinates promoter activity of the reporter plasmid in C2C12 and 3T3L1 cell lines.
Fig. 4 is KLF15 and PPAR γ Binding site for transcription factor mutation analysis.
Specific implementation mode
Embodiment below does not limit the present invention convenient for being better understood from the present invention.Experiment in following embodiments
Method is unless otherwise specified conventional method.Test material as used in the following examples is unless otherwise specified city
It sells.
Embodiment 1
One, the uciferase activity analysis of FATP1 gene promoters
1, promoter primer designs
According to 5 ' UTR sequence of FATP1 genes in GeneBank, the transcription initiation site identified with 5 '-RACE leads to for+1
Cross Clone Manage Software for Design gene specific primers：
Sense primer is Forward Primer F1:5′-CTACTGTGGTGGGCACTTG-3′
Downstream primer is Reverse Primer R1：5′-TTGTTCCCTGGCTGACCTGGAG-3′.
PCR amplification is carried out by template of cow genome group DNA, target fragment is total from promoter sequence -1856 to+190
Count 2046bp.1.2 μ L (100ng) DNA profilings, 0.4 μ L KOD-Plus-Ver, 2.0 (1U/ are sequentially added in 200 μ LPCR pipes
μL)、2μL10×PCR Buffer、2μLdNTP(2mmol/L)、0.8μL MgSO4(25mol/L)、0.6μL
ForwardPrimer F1(10μM)、0.6μL Reverse Primer R1(10μM)、12.4μLddH2O amounts to 20 μ L, whirlpool
It is put into PCR instrument and is reacted after rotation concussion centrifugation, reaction condition is as follows：95 DEG C of 5min of pre-degeneration are denaturalized 97 DEG C of 20s, annealing
63.5 DEG C of 30s extend 72 DEG C of 126s, 35 cycles, last 72 DEG C of 8min.
2, the recycling of promoter amplified fragments, connection and conversion
PCR product is detected through 0.8% agarose gel electrophoresis, target fragment 2046bp, according to DNA plastic recovery kits
Specification operates, and carries out gel extraction target fragment.Recycle target fragment with16 DEG C of Simple carriers connect overnight
It connects, then conversion DH-5 α competent cells, after 37 DEG C of inversion culture 16h in picking monoclonal to centrifuge tube, cultivates 12-16h.
3, plasmid is extracted
Numerous Escherichia coli will be expanded in step 2 centrifuge tube and carry out bacterium solution PCR amplification, identified positive colony bacterium solution send Nanjing
Jin Sirui is sequenced, and sequencing result is as follows：
ctactgtggtgggcacttgaacttgcctgagagaaaccaccccctcccccacagcgctcatgttctatttatagcgc
aattcccgagctgtgatggtccatttattttagttcaacctatgcttcacttctctcgttcgtccagaaagttctga
taattccctctgctgaggcctgtggttacctggggaaaggagacacctgtctttctgtgtcccccgcctgttcattc
cccagatgagaagcctccacgaaactttgtgcatgatgccctgggaggtgatcagccagaggacaaccaatgtgcag
aggtgagaggtgccccacccaggatcaccccactggagaggctcagagcctgcttgaccctgaagcttgagttctgc
tccccttgtccaggtctcagttgccccttctgtgaaatggggacaggactgttccttcaccggtcatctggaaccct
ggagaattgctatgtttttggaactgggaattattcagatcttaggacgggaatgctgccctgtgttgtgtgatgtc
cctgggggtgtgtggaagcccttatgtaaagagacaaccaaagggcttcccaggtggcgctagtggtaaagaacctg
cttgccaatgctggagacgtaagagaggcacaggaaatgtaagagacgtgggttcgttccctgggtccgatgcctgg
gtcgggaagatcccctggaggacagcacagcaattcagtccagtattcttgcctggactatcccatggacagaggag
cctggcaggctacagtctgtagtgttgcaaagagttggacacaactgaagcgacttagcagcagcagcaggggtaaa
aaaagaacactgctcactgaaaacagcttgggtcagtgctagggaaagggtccctccactggaggagtactggcacc
atctcaagaaaaggtgggtctctagagccgttaggacttggggattgctgactgaagacccagaagagtgtctccag
tagccacgggaggagttagaagagattgttatgtggcatatgggatcttaagttgctccaccagggatgaaccctgt
ggaagcacagaatcttaataaccactggaccaccagggaagtcccatgtgtactactggtatttagccagggctcag
taaacttggaaagatggggggaagaggagagagaagaaggtggaagaagtcgggaggagggaaggccacaggatggg
aggagaaagggaagaggagtgggtggaggaggagaaggtgcacaacgcgggaagtggaggaaggctaagggaggggg
cagaaaacttgaagaagggaaggagaatgggggaggggggtgggggggaatcagggcaagatggagaagagggagaa
gtttaaggaagactgaaaggggaggaagggcagcaagctctctgcccacatttgtgaaagaatgaatctcaactcca
gctagtgcgacagtgtcaagcctcagtttctttacctgtaaaatggggtgatcacaatagcgctatctggaaggaaa
agagtagctgctatagtttctgggcattcctgatccctagctgagaaagtcggggaccagtggctggctctgggcct
gtgctccggatctgcctcctcgtccccatttcacccaccctaggcagcccgcagggcccaatggcagcatgtgtggg
ggagtgcctgcccagtcctgtcctctaaatggtgctggaagcagacgctggctgcctcccaaggagagagctgagaa
ggtcggccaagcaggaaagaaacaagcaggggtgggataggcaggggggcagaggtagggggagcttggagagaggg
ttccaagggagaagcccacatgggcacaatgcaatcacagcaggccccatgagacggcagaaccagaagccccaagg
ggaggtcctctgtcttggcctcactgtcggtgtccgcctcctgcctgagcttctgggagcccacgaccgagcagcca
aagcctgaggatccgtgagcggctccaggtcagccagggaacaa
Wherein, the base of overstriking is+1.
Middle amount kit extraction plasmid is carried with reference to Omega companies operational manual is small for correct bacterium solution is sequenced, and is obtainedAnd preserve bacterium solution.
4, FATP1 genes 5 ' hold promoter series of deletions luciferase reporter vector structure
(1) design of primers
It is prepared with step 3Bacterium solution is template, is started using PCR method amplification FATP1
Subfamily deletion fragment.Using Clone Manage Software for Design special primers, addition SacI is held in upstream and downstream primer 5 ' respectively
With XhoI restriction enzyme sites, PCR primer is shown in Table 1.
1 N of FATP1 gene promoter deletion fragment amplification of table
(2) 5 ' end promoter series of deletions fragment amplifications
12.6 μ LddH are sequentially added in 200 μ LPCR pipes2O, 0.6 μ L sense primers (10 μM), 0.6 μ L downstream primers
(10μM)、0.8μL MgSO4(25mol/L)、2μLdNTP(2mmol/L)、2μL10×PCR Buffer、0.4μLKOD-Plus-
Ver 2.0 (1U/ μ L) and 1.0 μ L template DNAs (Bacterium solution), amount to 20 μ L.It is vortexed after concussion centrifuges and puts
Enter in PCR instrument and reacted, reaction condition is as follows：95 DEG C of 5min of pre-degeneration are denaturalized 97 DEG C of 20s, and anneal 63.5 DEG C of 30s, extends
72 DEG C of 126s, 35 cycles, last 72 DEG C of 8min, PCR product are detected with 0.8% agarose gel electrophoresis.
(3) series of deletions luciferase reporter gene vector construction
The serial PCR product that step (2) amplification obtains is purified respectively, then uses SacI+XhoI double digestions respectively
Digestion, the pGL3-Basic carriers then crossed with double digestion are attached, convert after carry out the identification of bacterium solution PCR amplification, be sequenced.Into
And obtain ox FATP1 genes 5 ' and hold promoter series of deletions reporter plasmid, it is respectively designated as pGL-1856/+190 (P1), pGL-
1558/+190(P2)、pGL-1261/+190(P3)、pGL-955/+190(P4)、pGL-640/+190(P5)、pGL-387/+
190(P6)、pGL-96/+190(P7)。
Wherein, use SacI+XhoI double digested respectively PCR product after purification, the pGL3- then crossed with double digestion
Basic carriers are attached, convert after carry out the detailed steps of bacterium solution PCR positive clone identifications and be：
By the double digestion mirror of pcr amplification product and reporter gene pGL3-Basic progress Sac I and Xho I after purification
It is fixed.DdH is sequentially added in reaction system2O、10×Quickcut Green Buffer、QuickcutXho Ⅰ、Quickcut
Sac I, pcr amplification product and pGL3-Basic carriers, concussion mixing centrifugation, 37 DEG C of water-bath 3h carry out digestion reaction, reaction
System is shown in Table 2.
2 digestion system of table
0.8% agarose gel electrophoresis of the digestion products of above-mentioned carrier is detected, then gel extraction purifies.Reference
4 ligase specification of NEB company's Ts sequentially adds pcr amplification product, 2 μ L enzymes after 5 μ L (50ng) digestions in the reaction system
Rear pGL3-Basic carriers, 1 μ L T4DNA, 1 μ L10 × T4DNA Ligase Buffer, moisturizing are cut to 10 μ L, reaction system is shown in
Table 3.It is vortexed after concussion mixing centrifugation, 16 DEG C of connections overnight.
3 linked system of table
Connection product is converted, positive colony bacterium solution PCR identifications, send sequencing, detailed step is the same as step 2 and step 3.
Plasmid is extracted to the correct bacterium solution of sequencing result, obtains FATP1 line fluorescent element enzyme Reporter gene vectors.
Plasmid is extracted to carry out according to Omega companies extraction plasmid kit specification：
(1) correct bacterium solution will be sequenced to be inoculated into 15mL ammonia benzyl culture mediums, 37 DEG C of shaking table culture 16h；
(2) bacterium is collected with 10000 × g centrifugations 1min at room temperature；
(3) culture medium is discarded, 500 μ L Solution I/RNase mixed liquors are added, vortex oscillation makes cell suspend completely；
(4) 500 μ L Solution II are added into resuspension mixed liquor, gently overturn mixing 4-6 times.This operation avoids play
Strong mixing lysate, and cracking reaction does not exceed 5min；
(5) 700 μ L Solution III are added, it is mild reverse for several times to formation white flock precipitate；
(6) at room temperature, 10min is centrifuged with >=12000 × g；
(7) transfer supernatant is centrifuged with 10000 × g at room temperature to being cased in the HiBind DNA columns of 2ml collecting pipes
1min removes the liquid of collecting pipe；
(8) pillar is reinstalled in collecting pipe, 500 μ LHB Buffer is added, centrifuged by above-mentioned condition, go filtrate；
(9) pillar is reinstalled in collecting pipe, 700 μ L DNA Wash Buffer is added, centrifuged, abandon by above-mentioned condition
Go filtrate；
(10) filtrate is discarded, it is primary to repeat the operation of the 9th step；
(11) filtrate is discarded, pillar is reinstalled collecting pipe, 12000 × g centrifuges void column 2min to dry column matrix；
(12) pillar on clean 1.5mL centrifuge tubes, 80-100 μ L Elution Buffer, -20 DEG C of guarantors are added
It deposits spare.
5, cell culture and the end of ox FATP1 genes 5 ' start the transfection of subfamily reporter plasmid
Mouse C2C12 cell lines and the culture of 3T3-L1 cell lines are in the culture medium comprising 90%DMEM and 10% fetal calf serum
In, the good cell of growth conditions is seeded in the day before transfection in 24 orifice plates, is 1 × 10 per hole inoculum density5A cell,
Then in 37 DEG C, 5%CO2It is cultivated in incubator, when cell density reaches 70~80%, ox FATP1 genes 5 ' is held and are started
Subfamily luciferase reporter gene carrier (pGL-1856/+190 (P1), pGL-1558/+190 (P2), pGL-1261/+190
(P3), pGL-955/+190 (P4), pGL-640/+190 (P5), pGL-387/+190 (P6) and pGL-96/+190 (P7)) transfection
Cell.Use pRL-TK as internal reference plasmid while transfection, pGL3-Basic makees as negative control plasmids, pGL3-Control
For positive control plasmid.Up time transfection procedure carries out plasmid transfection according to Roche X-treme GENE HP operating instructions, turns every time
Three multiple holes are contaminated, specific transfection procedure is as follows：
(1) preparation of A liquid：50μLopti-MEM+1.6μLX-treme GENE HP；
(2) preparation of B liquid：50μLopti-MEM+10ng(pRL-TK)+800ng(pGL-3)；
(3) after A liquid preparation 5min 20min is stored at room temperature with B liquid mixings；
(4) 100 μ L mixed liquors are uniformly added dropwise in every hole, luciferase assays after 48h.
7, uciferase activity is analyzed
(1) passive 1 × PLB of lysate is prepared：5 × Passive Lysis Buffer (PLB) of 1 times of volume are added to 4
It in the distilled water of times volume, is uniformly mixed, 4 DEG C preserve (≤1 month)；
(2) LAR II is prepared：With II solution solution freeze-dried powder Luciferase Assay of Luciferase Assay Buffer
Substrate, -20 DEG C preserve (≤1 year)；
(3) a certain amount of 1 × Stop＆ is configuredReagent：By a certain amount of 50 × Stop＆Substrate adds
Enter to required Stop＆In Buffer, make final concentration of 1 times of concentration (- 20 DEG C preserve 15 days)；
(4) culture medium after the plasmid transfection that aspiration step 6 obtains in cell cleans the cell of culture with 1 × PBS, goes
Fall cleaning solution；
(5) 80 μ L of passive lysate are added in every hole, 200 turns/min of shaking table shakes 15min at room temperature；
(6) it takes in the 20 μ L to 96 orifice plates of lysate that step (5) obtains, the detection examination of 50 μ L firefly luciferases is added
Fluorescence intensity level (A values) is read in agent after mixing on multi-function microplate reader；
(7) 50 μ L renilla luciferase detection reagents are subsequently added into, it is strong that fluorescence is read on multi-function microplate reader after mixing
Angle value (B values)；
(8) relative transcriptional activity of promoter series of deletions segment is calculated with A values/B values.
Two, statistical analysis
Experimental result is indicated with average ± standard deviation, and number is tested using SPSS16.0 statistical analysis software analyzing processings
According to, more comparison among groups are carried out using one-way analysis of variance method, and comparison among groups use LSD-t methods of inspection, 0.05 tables of * p ＜
Show that significant difference, * * p ＜ 0.01 indicate that difference is extremely notable.
Three, result
1, FATP1 gene promoters expand
Using Qinchuan Cattle poba gene group DNA as template, promoter special primer, PCR amplification FATP1 gene promoters are designed
Sequence obtains the PCR product (Fig. 1) of about 2000bp, connectionBlast comparisons are carried out after the sequencing of Simple carriers.
The results show that clone's FATP1 promoter sequences are consistent with the registered ends FATP15 ' promoter sequence in ncbi database, table
Bright successful clone ox FATP1 gene promoter sequences, and submit sequence to ncbi database (GenBankNo.KU215705).
Fig. 1 is FATP1 gene promoter amplifications；Wherein, M:DL2000DNA Marker；1, FATP1 gene promoter
Sub- PCR product.
2, FATP1 gene promoters serial deletions luciferase reporter vector is built
To determine ox FATP1 gene core promoters region, withBacterium solution is template, PCR amplification
Promoter FATP1 genes 5 ' hold promoter series of deletions segment, through glue recovery purifying, double digestion, connection pGL3-Basic carriers,
FATP1 promoter series of deletions luciferase reportings are built after converting DH5 α competence, the screening of bacterium solution PCR positive colonies, sequencing
Gene plasmid.Obtain recombination deficient plasmid P1, P2, P3, P4, P5, P6, P7.By P1, P2, P3, P4, P5, P6 and P7 through SacI and
It after XhoI double digestions, is detected with 0.8% agarose gel electrophoresis and two specific bands occurs, wherein a treaty 4.8kb, another
Item is respectively 2046bp, 1748bp, 1451bp, 1145bp, 830bp, 577bp, 286bp (Fig. 2).It is proved through forward and reverse sequencing
The success of FATP1 gene promoter series of deletions luciferase reporter gene vector constructions.
Fig. 2 identifies for pGL-FATP1 different fragments Sac I and Xho I double digestions；Wherein, M:DL2000DNA Marker；
P1：pGL-1856/+190；P2：pGL-1558/+190；P3：pGL-1261/+190；P4：pGL-955/+190；P5：pGL-
640/+190；P6：pGL-387/+190；P7：pGL-96/+190.
3, FATP1 gene core promoters region is determined
The gene promoter containing FATP1 5 ' built is held to the firefly luciferase report of series of deletions segment recombinant plasmid
Expression vector, Renilla luciferase reporter genophore pRL-TK are accused as internal reference difference cotransfection 3T3L-1 and C2C12
Cell line, hollow carrier pGL-Basic is as negative control, pGL-Control carriers as positive control.Cell culture
After 48h, the relative activity of luciferase is detected.As shown in figure 3, in 3T3L1 and C2C12 cell lines, pGL-1856/+190 matter
Grain promoter relative activity is 5 and 22 times of negative control pGL-Basic, illustrates that we are at clened cows FATP1 gene promoters
One functional promoter.
Fig. 3 is that gene promoter recombinates promoter activity of the reporter plasmid in C2C12 and 3T3L1 cell lines.
A series of FATP1 genes 5 ' hold the promoter activity of deleted promoter fragments recombinant plasmid reporter gene expression carrier
Trend is similar in two kinds of cell line.When promoter is from -1856 missings to -1558, promoter is lived in C2C12 cell lines
Property increases 9.3 times, illustrates -1856 that have important negative regulatory element to be present in FATP1 promoters Dao -1558；But
When from -1558 missings to -96, promoter activity changes there is no apparent in two kinds of cell line, illustrates at -1558
To -96 and important controlling element is not present；Activity of the pGL-96/+190 promoters in C2C12 and 3T3L1 is pGL-
13 and 11 times of baisc illustrate that ox FATP1 gene core promoters region is located at (p in -96 to+190 regions<0.01).
Four, it determines and maintains the active controlling element of FATP1 gene promoter transcriptions
1, FATP1 gene promoters rite-directed mutagenesis is tested
(1) rite-directed mutagenesis primer designs
It is predicted according to online software, to ox FATP1 gene promoter transcription initiation sites upstream -96bp~regions+196bp
PPAR γ (- 38bp to-16bp), KLF15 (- 99bp to-77bp) transcription factor key binding sites are (in table 4 under primer
Scribing line is the base position of mutation) carry out rite-directed mutagenesis, referenceLightningSite-Directed
Mutagenesis Kit kit specifications, which design, includes the primer of mutant nucleotide sequence, wherein primer length between 25~45bp,
Value >=78 DEG C Tm, G/C content must be more than 40%, 3 ' end of primer and be necessarily base C or G, and mutational site should draw
Among object sequence, primer particular sequence is shown in Table 4.
The primer of 4 mutant plasmid of table
(2) rite-directed mutagenesis reaction system
According toLightningSite-Directed Mutagenesis Kit kit specifications,
5 μ L 10 × reaction buffer, 5 μ L (25ng) plasmids (pGL-387/190), 1.25 μ L are sequentially added in reaction system
(125ng) forward primer, 1.25 μ L (125ng) reverse primers, 1 μ LdNTP mix, 1.5 μ LQuickSolution reagent,
34μLddH2O、1Lightning Enzyme are put into PCR instrument after the concussion centrifugation that is vortexed and are reacted,
Reaction condition is as follows：95 DEG C of 2min of pre-degeneration are denaturalized 97 DEG C of 20s, and anneal 60 DEG C of 10s, extend 68 DEG C of 30s, and 18 recycle, finally
68℃5min.After PCR, ice bath 2min is subsequently added into 1 μ LDpn ì enzymes, 37 DEG C of digestion 10min.
(3) it converts and is sequenced
PCR product is converted in super competence XL10-Gold bacteriums.Specific steps are shown in second part, sequencing
Bacterium is shaken after correct, -20 DEG C of plasmid of extraction saves backup.
(4) cell culture and transient transfection
C2C12 cell culture is in containing the high sugared culture solutions of 10% fetal calf serum+90%DMEM, in 37 DEG C of 5%CO2Cell
Secondary culture is carried out in incubator.For 24 hours by cell inoculation in 24 orifice plates before transfection.Using Roche reagent (X-tremeGENE
HP it) is transiently transfected, each plasmid turns 3 holes, and specific transfection method is identical as the 6th step of first part.What is transfected is same
When use pRL-TK as internal reference plasmid, pGL3-Basic is as negative control plasmids, and pGL3-Control is as positive control matter
Grain.
2, experimental result：KLF15 Binding site for transcription factor maintains FATP1 promoters basic transcription activity
By building series of deletions promoter fragment carrier, transfection C2C12 and 3T3L1 cells carry out uciferase activity point
After analysis, determine that promoter minimum active region is located in -96bp~regions+190bp, wherein being deposited between -96bp~+190bp
The transcription factor in the important regulating and controlling element of regulation and control FATP1 gene promoter activities, our follow-up work Main Analysis regions
Binding site.Find that there are multiple latent in FATP1 gene promoters region using Gemomatix and TFSEARCH online software analyses
Binding site for transcription factor, including Binding site for transcription factor such as PPAR γ, KLF15.
Further to study effect of these Binding site for transcription factor in maintaining FATP1 gene promoter activities, according to
Corresponding primer pair pGL-96/+190 plasmids are designed according to each Binding site for transcription factor and carry out rite-directed mutagenesis, and are transiently transfected
C2C12 cells.Experimental result is shown in Fig. 4.
Fig. 4 is the Assay of promoter activity after the mutation of KLF15 and PPAR γ Binding site for transcription factor.
The experimental results showed that：Rite-directed mutagenesis PPAR γ Binding site for transcription factor is to FATP1 promoter activities without notable shadow
It rings (p ＞ 0.05), after being mutated KLF15 Binding site for transcription factor, starts activity and decline 70% (p ＜ 0.01)；Illustrate that KLF15 turns
Record factor binding site is that the required regulation and control of ox FATP1 gene promoters basic transcription activity is maintained to make element.
Finally it should be noted that：The foregoing is only a preferred embodiment of the present invention, is not intended to restrict the invention,
Although the present invention is described in detail referring to the foregoing embodiments, for those skilled in the art, still may be used
With technical scheme described in the above embodiments is modified or equivalent replacement of some of the technical features.
All within the spirits and principles of the present invention, any modification, equivalent replacement, improvement and so on should be included in the present invention's
Within protection domain.
Sequence table
<110>Gansu Agriculture University
<120>The method of the identification regulation and control active controlling element of FATP1 gene promoter transcriptions
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 2046
<212> DNA
<213>Fatty acid transport protein 1 (Fatty acid transport protein 1)
<400> 1
ctactgtggt gggcacttga acttgcctga gagaaaccac cccctccccc acagcgctca 60
tgttctattt atagcgcaat tcccgagctg tgatggtcca tttattttag ttcaacctat 120
gcttcacttc tctcgttcgt ccagaaagtt ctgataattc cctctgctga ggcctgtggt 180
tacctgggga aaggagacac ctgtctttct gtgtcccccg cctgttcatt ccccagatga 240
gaagcctcca cgaaactttg tgcatgatgc cctgggaggt gatcagccag aggacaacca 300
atgtgcagag gtgagaggtg ccccacccag gatcacccca ctggagaggc tcagagcctg 360
cttgaccctg aagcttgagt tctgctcccc ttgtccaggt ctcagttgcc ccttctgtga 420
aatggggaca ggactgttcc ttcaccggtc atctggaacc ctggagaatt gctatgtttt 480
tggaactggg aattattcag atcttaggac gggaatgctg ccctgtgttg tgtgatgtcc 540
ctgggggtgt gtggaagccc ttatgtaaag agacaaccaa agggcttccc aggtggcgct 600
agtggtaaag aacctgcttg ccaatgctgg agacgtaaga gaggcacagg aaatgtaaga 660
gacgtgggtt cgttccctgg gtccgatgcc tgggtcggga agatcccctg gaggacagca 720
cagcaattca gtccagtatt cttgcctgga ctatcccatg gacagaggag cctggcaggc 780
tacagtctgt agtgttgcaa agagttggac acaactgaag cgacttagca gcagcagcag 840
gggtaaaaaa agaacactgc tcactgaaaa cagcttgggt cagtgctagg gaaagggtcc 900
ctccactgga ggagtactgg caccatctca agaaaaggtg ggtctctaga gccgttagga 960
cttggggatt gctgactgaa gacccagaag agtgtctcca gtagccacgg gaggagttag 1020
aagagattgt tatgtggcat atgggatctt aagttgctcc accagggatg aaccctgtgg 1080
aagcacagaa tcttaataac cactggacca ccagggaagt cccatgtgta ctactggtat 1140
ttagccaggg ctcagtaaac ttggaaagat ggggggaaga ggagagagaa gaaggtggaa 1200
gaagtcggga ggagggaagg ccacaggatg ggaggagaaa gggaagagga gtgggtggag 1260
gaggagaagg tgcacaacgc gggaagtgga ggaaggctaa gggagggggc agaaaacttg 1320
aagaagggaa ggagaatggg ggaggggggt gggggggaat cagggcaaga tggagaagag 1380
ggagaagttt aaggaagact gaaaggggag gaagggcagc aagctctctg cccacatttg 1440
tgaaagaatg aatctcaact ccagctagtg cgacagtgtc aagcctcagt ttctttacct 1500
gtaaaatggg gtgatcacaa tagcgctatc tggaaggaaa agagtagctg ctatagtttc 1560
tgggcattcc tgatccctag ctgagaaagt cggggaccag tggctggctc tgggcctgtg 1620
ctccggatct gcctcctcgt ccccatttca cccaccctag gcagcccgca gggcccaatg 1680
gcagcatgtg tgggggagtg cctgcccagt cctgtcctct aaatggtgct ggaagcagac 1740
gctggctgcc tcccaaggag agagctgaga aggtcggcca agcaggaaag aaacaagcag 1800
gggtgggata ggcagggggg cagaggtagg gggagcttgg agagagggtt ccaagggaga 1860
agcccacatg ggcacaatgc aatcacagca ggccccatga gacggcagaa ccagaagccc 1920
caaggggagg tcctctgtct tggcctcact gtcggtgtcc gcctcctgcc tgagcttctg 1980
ggagcccacg accgagcagc caaagcctga ggatccgtga gcggctccag gtcagccagg 2040
gaacaa 2046
Claims (7)
1. the method for the identification regulation and control active controlling element of FATP1 gene promoter transcriptions, includes the following steps：
(1) FATP1 gene promoter sequences are cloned；
(2) structure FATP1 genes 5 ' hold promoter series of deletions segment luciferase reporter vector；
(3) the FATP1 genes 5 ' of step (2) end promoter series of deletions segment luciferase reporter vector is transfected respectively
Cell carries out uciferase activity analysis, determines FATP1 gene core promoters active region；
(4) Binding site for transcription factor in analysis FATP1 gene core promoters active region, to Binding site for transcription factor
Rite-directed mutagenesis is carried out respectively, and regulation and control FATP1 gene promoters are determined according to the FATP1 gene promoter activity situations of change after mutation
The controlling element of sub- transcriptional activity：If after being mutated to Binding site for transcription factor, startup activity, which is remarkably decreased, illustrates the transcription
Factor binding site is the regulation and control active controlling element of FATP1 gene promoter transcriptions.
2. according to the method described in claim 1, it is characterized in that：Clone's FATP1 gene promoter sequences, structure
FATP1 gene promoter series of deletions segment luciferase reporter vectors include the following steps：
(1) according to 5 ' UTR sequence of FATP1 genes, the transcription initiation site identified with 5 '-RACE designs FATP1 genes for+1
Promoter specific primer carries out PCR amplification to FATP1 gene promoters, target fragment be from promoter sequence -1856 to
+ 190；
(2) pcr amplification product glue is recycled, and with19-T Simple carriers connect, and transformed competence colibacillus cell is trained
It supports, positive colony bacterium solution is sequenced, plasmid then is extracted to the correct bacterium solution of sequencing result；Design primer and with above-mentioned survey
The correct bacterium solution of sequence is template, and amplification FATP1 genes 5 ' hold promoter series of deletions segment；
(3) promoter series of deletions segment and luciferase reporter vector is held to distinguish enzyme the FATP1 genes 5 ' of step (2) amplification
It is connected after cutting, connection product is converted, positive colony bacterium solution is sequenced, then the correct bacterium solution of sequencing result is carried
Plasmid is taken, luciferase reporter vector is obtained：pGL-1856/+190(P1)、pGL-1558/+190(P2)、pGL-1261/+190
(P3)、pGL-955/+190(P4)、pGL-640/+190(P5)、pGL-387/+190(P6)、pGL-96/+190(P7)。
3. according to the method described in claim 2, it is characterized in that：Specific primer is described in step (1)：
Sense primer is F1:5′-CTACTGTGGTGGGCACTTG-3′；
Downstream primer is R1：5′-TTGTTCCCTGGCTGACCTGGAG-3′.
4. according to claim 1-3 any one of them methods, it is characterised in that：Series FATP1 genes described in step (2) 5 '
End promoter sequence deletion fragment luciferase reporter vector be：pGL-1856/+190(P1)、pGL-1558/+190(P2)、
pGL-1261/+190(P3)、pGL-955/+190(P4)、pGL-640/+190(P5)、pGL-387/+190(P6)、pGL-96/+
190(P7)。
5. according to claim 1-3 any one of them methods, it is characterised in that：It opens at the end of FATP1 genes 5 ' described in step (3)
Mover series of deletions segment luciferase reporter vector transfectional cell is transfected C2C12 cell lines and 3T3-L1 cell lines.
6. according to claim 1-3 any one of them methods, it is characterised in that：It opens at the end of FATP1 genes 5 ' described in step (3)
Use pRL-TK as internal reference plasmid, pGL3- while the luciferase reporter vector transfectional cell of mover series of deletions segment
Basic is as negative control plasmids, and pGL3-Control is as positive control plasmid.
Application of the 7.KLF15 Binding site for transcription factor in maintaining FATP1 gene promoter basic transcription activity.
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