CN105969768A - KLF7 gene promoter as well as activity and application thereof - Google Patents
KLF7 gene promoter as well as activity and application thereof Download PDFInfo
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- CN105969768A CN105969768A CN201610306116.7A CN201610306116A CN105969768A CN 105969768 A CN105969768 A CN 105969768A CN 201610306116 A CN201610306116 A CN 201610306116A CN 105969768 A CN105969768 A CN 105969768A
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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- C12N15/09—Recombinant DNA-technology
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Abstract
The invention provides a KLF7 gene promoter as well as activity and application thereof. The sequence of the KLF7 gene promoter is shown as a sequence table SEQ ID No.1. The KLF7 gene promoter proves that the chicken KLF7 gene promoter has a capability of promoting gene expression in a DF1 cell and a regulation and control effect on the activity of the promoter by two transcription factors is researched; a novel research tool is provided for application of an expression regulation and control mechanism of a KLF7 gene and the KLF7 to chicken genetic breeding; references are provided for screening new broiler low-fat genes for high-quality low-fat broiler breeding and the KLF7 gene promoter can be directly used for detecting complicated KLF7 transcription regulation and control.
Description
Technical field
The invention belongs to gene engineering technology field, be specifically related to a kind of KLF7 gene promoter and activity thereof and application.
Background technology
Kr ü ppel-like factor 7 (KLF7), is again ubiquitous Kruppel-like factor (UKLF),
It is that a c-terminus has 3 Cys2-His2The transcription factor of zinc fingers, belongs to transcription factor sp-KLFs family.NCBI
Gene database information retrieval shows, KLF7 gene is present in Rana nigromaculata, chicken, mice and people in 100 interior many animals bodies.
Expression analysis shows, KLF7 wide expression is in adult, mammal and the Various Tissues of birds.Mammal functional study table
Bright, the differentiation potential of KLF7 regulation and control stem cell, promote nervous system development, and be human obesity and type 2 diabetes mellitus and blood
The disease pathogenetic important regulating and controlling factor.
Broiler is the most common poultry of artificial rearing, has important economic worth.Through breeder's more than 90 year
Unremitting effort, at present, the daily gain of commercial generation broiler chicken and feed conversion rate have all reached the highest level.But, due to
Selection excessive to growth traits during long-term breeding, causes present commercial generation broiler chicken to occur in that stomach fat accumulation too much
Problem.Stomach fat is too much accumulated not only affects broiler production efficiency, causes feed waste, and can cause broiler sudden death syndrome and
Pollute environment.Cultivating high-quality low fat broiler is to control the effective means that broiler stomach fat is too much accumulated.
The research of multiple broiler colonies shows, of chicken KLF7 coding region (Coding DNA Sequence, CDS) is same
Gene pleiomorphism (the SNP:c.A141G of justice sudden change；AFK10495.1:p.Pro47Pro) with broiler abdominal fat content and blood plasma
Middle very low density lipoprotein (VLDL) (very low density lipoprotein, VLDL) horizontal significant correlation, and this site can
The protein expression level of KLF7 can be acted on by codon preference.High and low fat system broiler KLF7 tissue expression analysis of spectrum shows
Show, KLF7 the liver of 7 week old high and low fat broiler, duodenum, jejunum, ileum, chest muscle, lower limb flesh, stomach fat, heart, spleen,
Kidney, pancreas, glandular stomach, brain and testis all have expression.Analyze the expression water of KLF7 in 1-12 week old broiler abdominal adipose tissue
Flat discovery, the mrna expression level of low fat system broiler abdominal adipose tissue KLF7 is significantly higher than high fat system broiler abdominal adipose tissue
The mrna expression level of KLF7.These researchs show, KLF7 is the important controlling gene of broiler stomach fat growth promoter, research
KLF7 gene expression and regulation mechanism in broiler abdominal adipose tissue growth and development process contributes to disclosing broiler abdomen further
The lipopectic molecular mechanism in portion, and accelerate to be applied to KLF7 the process of low fat broiler breeding.Promoter is genes of interest
Switch, controls the expression of gene, therefore studies impact and the Control factors of a gene promoter, is to understand this gene action
Mechanism and an important step of regulation factor, therefore interval and characteristic for its promoter of gene studies just seem outstanding
For important.
Functional study shows, KLF7 all has expression, and KLF7mRNA in chicken PECTORAL LIMB SKELETON and mature fat cell
Expression presents with the differentiation of adipose cell after falling before and rises the trend reduced again；Process LAN KLF7 promotes fat before chicken
Cell proliferation, suppression chicken PECTORAL LIMB SKELETON differentiation.Chicken KLF7 is right likely via the expression performance of directly suppression C/EBP α gene
The regulating and controlling effect of chicken lipocyte proliferation differentiation.Although KLF7 stomach fat deposit in effect it is verified that.But, mesh
The regulatory mechanism that front KLF7 expresses is unclear, and the research to the promoter of chicken KLF7 gene has no report.Determine that chicken KLF7 opens
Mover, the most significant for gene functional research and low fat broiler breeding.
Summary of the invention
It is an object of the invention to provide a kind of KLF7 gene promoter and activity thereof and application, it is possible to be directly used in detection
Complicated KLF7 transcriptional control.
The purpose of the present invention is realized by following technology: a kind of KLF7 gene promoter, sequence such as sequence table SEQ ID
Shown in No.1.
The present invention also has a following technical characteristic:
1, a kind of KLF7 gene promoter as above, at the promoter activity that DF1 cells show goes out.
2, a kind of KLF7 gene promoter as above, transcription factor Gata2 and the Gata3 suppression to promoter activity
Effect.
3, a kind of KLF7 gene promoter as above, the application in the genetic breeding of chicken.
4, a kind of KLF7 gene promoter as above, at the promoter activity that DF1 cells show goes out, with recombinant vector
PGL3-Basic-KLF7p transfects DF1 cell, and recombinant vector sequence is as shown in sequence table SEQ ID No.2.
5, a kind of primer pair synthesizing KLF7 gene promoter, forward primer KLF7pF: sequence such as sequence table SEQ ID
Shown in No.3；Downstream primer KLF7pR: sequence is as shown in sequence table SEQ ID No.4.
The present invention is used in gene function field, it is possible to the medicine expressed for screening regulation and control KLF7 is expressed with disclosing KLF7
Transcription regulation mechanism；It is used in broiler production field, can be at cellular and molecular level for screening high-quality low fat broiler, Yi Jiyong
In the feeding and management scheme optimizing high quality meat chicken.The invention discloses transcription factor Gata2 and Gata3 KLF7 gene is opened simultaneously
The regulating and controlling effect of promoter activity, is applied to gene function field for the present invention and screens new broiler low fat gene for excellent
Matter low fat broiler breeding provides reference.
Accompanying drawing explanation
Fig. 1 is that the double digestion of recombiant plasmid pGL3-Basic-KLF7p is identified.
Fig. 2 is pGL3-Basic-KLF7p promoter function analysis result.
Fig. 3 is KLF7 gene promoter area partial sequence Transcription Factor Binding Sites Prediction result.
Fig. 4 is pCMV-Myc-Gata2 and pCMV-Myc-Gata3 expressed fusion protein qualification result.
Fig. 5 is the qualification result that KLF7 gene promoter activity is affected by Gata2 and Gata3.
Detailed description of the invention
The experimental technique used in the following example if no special instructions, is conventional method.
Material used in the following example, reagent etc., if no special instructions, the most commercially obtain.
Embodiment 1
The preparation of KLF7 gene promoter
1, chicken extracting genome DNA
(1) take 7 week old AA broiler of normal cultivation, wing venous blood collection 1mL, install to be previously added by the Sanguis Gallus domesticus of collection
10L anticoagulant is (such as 0.5mol/L Na2EDTA) in 1.5mLEP pipe, mixing of turning upside down.
(2) take the anticoagulant Sanguis Gallus domesticus of 20 μ L in a new 1.5mLEP pipe, add 445 μ L 1 × SET (100mM Tris-
HCl PH7.5,100m M EDTA, after autoclaving, the SDS adding 1%), 10 μ L PK (10mg/mL) and 25 μ LSDS (10%),
Fully mixing, digests 12h by 55 DEG C.
(3) add the 500 μ saturated phenol of L Tris, fully mix, be put in shaking table the 10min that turns upside down.
(4) 12000r/m is centrifuged 10min, sucts and manages to new EP clearly.
(5) add 400 μ L phenol chloroform isoamyl alcohol (volume ratio 24:23:1), fully mix, be put in shaking table and turn upside down
10min。
(6) 12000r/m is centrifuged 10min, sucts layer and manages to new EP.
(7) add 400 μ L chloroforms: isoamyl alcohol (volume ratio 23:1), fully mix, be put in shaking table the 10min that turns upside down.
(8) 12000r/m is centrifuged 10min, sucts layer and manages to new EP.
(9) 800 μ L (2 times of volumes) dehydrated alcohol (-20 DEG C of pre-coolings) is added, concussion, it is seen that white flock precipitate.
(10) 8000r/m is centrifuged 10min, abandons liquid, portion's white precipitate of keeping on file.
(11) 1mL 70% ethanol (-20 DEG C of pre-coolings) is added, concussion, wash white precipitate DNA.
(12) 8000r/m is centrifuged 10min, abandons liquid, portion's white precipitate of keeping on file；EP pipe is put upside down on filter paper, to tube wall
Aneroid pearl.
(13) adding the 200 aseptic TE of μ L, 55 DEG C of water-baths are dissolved.
2, PCR expands KLF7 gene promoter
Obtaining KLF7 genome sequence by UCSC genome database, 3 kinds of KLF7 in conjunction with ncbi database prediction turn
Recording this information, at 3 kinds of KLF7 transcript near transcriptional start sites region design primer KLF7pF and KLF7pR of prediction, utilizing should
Primer clone's KLF7 prediction transcriptional start site upstream about 2000bp sequence is (relative to sequence X M_004942644.2 first
Base ,-2026/+171).
Primer sequence is as follows:
KLF7pF:5 '-CGGGGTACCCGAATAAACCTAAGCATGAGCAATC-3 '
KLF7pR:5 '-GGAAGATCTCAACCAATGATAGATCCAGCGTCC-3 '
The chicken genomic DNA extracted in 1 is as template, KLF7pF and KLF7pR is that primer carries out PCR amplification.By mixture
94 DEG C of denaturations 5min；94 DEG C of degeneration 30s, 65 DEG C of annealing 25s, 72 DEG C extend 2min 30s, totally 36 circulations；72 DEG C of ends prolong
Stretching 10min, 4 DEG C terminate reaction.
Mixture system is as follows:
PCR amplification condition is: 94 DEG C of denaturations 5min；94 DEG C of degeneration 30s, 65 DEG C of annealing 25s, 72 DEG C extend 2min
30s, totally 36 circulations；72 DEG C extend 10min eventually, and 4 DEG C terminate reaction.
PCR primer is carried out agarose gel electrophoresis, reclaims purpose band.Genes of interest is connected to pMD-18T carrier
(Takara), on, product transform E. Coli DH5 α, Amp are connected+Resistance screening cultivates monoclonal, and monoclonal is inoculated in LB liquid
Body culture medium expands, and extracts plasmid, is checked order by plasmid, by the named pMD-18T-KLF7p of plasmid correct for order-checking.
3, KLF7 promoter luciferase reporting genophore builds
Respectively with pMD-18T-KLF7p carrier and pGL3-Basic carrier for enzyme action substrate, Kpn I and Bgl II is utilized to carry out
Double digestion, enzyme action system is as follows:
Enzyme action condition is: 37 DEG C of enzyme action 1h.
Digestion products is identified through 1% agarose gel electrophoresis and uses AXYGEN gel purification kits to reclaim.
Utilize T4DNA ligase, connect genes of interest fragment KLF7p and skeleton carrier fragment pGL3-Basic, build pGL3-Basic-
KLF7p carrier.Connect product transform E. Coli DH5 α, Amp+Resistance screening is cultivated, and monoclonal is inoculated in by picking monoclonal
LB fluid medium amplification cultivation, extracts plasmid, utilizes Kpn I and Bgl II to carry out double digestion qualification, and result is as shown in Figure 1.Will
The plasmid obtained checks order, and check order the named pGL3-Basic-KLF7p of correct plasmid.
Embodiment 2
KLF7 gene promoter transcription activity analysis
1, KLF7 gene promoter activity checking
Being inoculated in 12 porocyte culture plates by DF1 cell good for growth conditions, inoculum density is 5 × 104Individual/hole,
Cultivate after 24h, according to Fugene HD (promega) transfection reagent description, pGL3-Basic-KLF7p is transfected into DF1 cell
In, every hole transfects 1 μ g plasmid, and transfection pGL3-Basic-KLF7p is process group, transfects 3 holes, transfects pGL3-Basic
(empty vector, EV) is matched group, transfects 3 holes, and transfection pRL-TK (Promega) is internal reference, and every hole transfects 0.02 μ g,
Cell is received, according to Promega company after transfection 48hLuciferase Assay System description is carried out
Fluorescence activity measures.Result is as in figure 2 it is shown, show that the PGL3-Basic-KLF7p built has promoter activity.
2, the transcription factor prediction of regulation and control KLF7 gene promoter
Utilize TransFac data base that the KLF7 gene promoter sequence obtained is carried out transcription factor prediction, result such as figure
Shown in 3, show this sequence has multiple Binding site for transcription factor, such as Gata2, Gata3 Binding site for transcription factor.
3, transcription factor Gata2 and the checking of Gata3 vector expression albumen
Being inoculated in 6 porocyte culture plates by DF1 cell good for growth conditions, inoculum density is 105Individual/hole, cultivates
After 24h, the carrier for expression of eukaryon pCMV-Myc-respectively laboratory preserved according to Fugene HD (promega) description
Gata2 and pCMV-Myc-Gata3 is transfected in DF1 cell, and every hole transfects 2.0 μ g plasmids, receives cell after transfection 48h.Utilize
Can two carrier for expression of eukaryon of western blotting technical identification expressed fusion protein.Result as shown in Figure 4, shows
PCMV-Myc-Gata2 and pCMV-Myc-Gata3 can merge egg by successful expression chicken Gata2 and Gata3 in DF1 cell line
In vain.
4, the qualification that KLF7 gene promoter activity is affected by transcription factor Gata2 and Gata3
Being inoculated in 12 porocyte culture plates by DF1 cell good for growth conditions, inoculum density is 5 × 104Individual/hole,
After 24h, being transfected in DF1 cell by each group of plasmid according to Fugene HD (promega) description, often group repeats three holes, point
Organize as follows:
Cell is received, according to Promega company after transfection 48hLuciferase Assay System explanation
Book carries out fluorescence activity mensuration, and result is as it is shown in figure 5, show the promoter activity of process LAN Gata2 and Gata3 suppression KLF7.
Claims (6)
1. a KLF7 gene promoter, it is characterised in that sequence is as shown in sequence table SEQ ID No.1.
A kind of KLF7 gene promoter the most according to claim 1, it is characterised in that in the startup that DF1 cells show goes out
Son activity.
A kind of KLF7 gene promoter the most according to claim 1, it is characterised in that transcription factor Gata2 and Gata3 pair
The inhibitory action of promoter activity.
A kind of KLF7 gene promoter the most according to claim 1, it is characterised in that answering in the genetic breeding of chicken
With.
A kind of KLF7 gene promoter the most according to claim 1, it is characterised in that in the startup that DF1 cells show goes out
Son activity, transfects DF1 cell, recombinant vector sequence such as sequence table SEQ ID No.2 with recombinant vector pGL3-Basic-KLF7p
Shown in.
6. the primer pair synthesizing KLF7 gene promoter, it is characterised in that forward primer KLF7pF: sequence such as sequence table
Shown in SEQ ID No.3；Downstream primer KLF7pR: sequence is as shown in sequence table SEQ ID No.4.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111500590A (en) * | 2020-05-11 | 2020-08-07 | 石河子大学 | Application of transcription factor GATA2 for regulating formation of chicken fat cells |
| CN112662673A (en) * | 2021-01-08 | 2021-04-16 | 石河子大学 | Human KLF7 gene promoter as well as construction method and application thereof |
| CN115287286A (en) * | 2022-06-24 | 2022-11-04 | 中国人民解放军军事科学院军事医学研究院 | Application of long-chain non-coding RNA lnc1267 in regulation of cell proliferation and survival |
-
2016
- 2016-05-10 CN CN201610306116.7A patent/CN105969768B/en not_active Expired - Fee Related
Non-Patent Citations (4)
| Title |
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| ZHANG Z等: "JX290203.1", 《GENBANK》 * |
| ZHI-WEI ZHANG等: "Cloning, tissue expression and polymorphisms of chicken Krüppel-like factor 7 gene", 《ANIMAL SCIENCE JOURNAL》 * |
| ZHIWEI ZHANG等: "Structure and phylogenetic analysis of chicken KLF7 gene", 《第五届全国生物信息学与系统生物学学术大会论文集》 * |
| 张志威等: "KLF 转录因子家族与脂肪细胞分化", 《中国生物化学与分子生物学报》 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111500590A (en) * | 2020-05-11 | 2020-08-07 | 石河子大学 | Application of transcription factor GATA2 for regulating formation of chicken fat cells |
| CN111500590B (en) * | 2020-05-11 | 2022-09-16 | 石河子大学 | Application of transcription factor GATA2 for regulating formation of chicken fat cells |
| CN112662673A (en) * | 2021-01-08 | 2021-04-16 | 石河子大学 | Human KLF7 gene promoter as well as construction method and application thereof |
| CN112662673B (en) * | 2021-01-08 | 2022-06-28 | 石河子大学 | Human KLF7 gene promoter as well as construction method and application thereof |
| WO2022148253A1 (en) * | 2021-01-08 | 2022-07-14 | 石河子大学 | Human klf7 gene promoter, construction method therefor and application thereof |
| CN115287286A (en) * | 2022-06-24 | 2022-11-04 | 中国人民解放军军事科学院军事医学研究院 | Application of long-chain non-coding RNA lnc1267 in regulation of cell proliferation and survival |
| CN115287286B (en) * | 2022-06-24 | 2023-03-07 | 中国人民解放军军事科学院军事医学研究院 | Application of long-chain non-coding RNA lnc1267 in regulation of cell proliferation and survival |
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