CN108715848A - Applications of the transcription factor CEBP α as the transcription factor of Kiss1 promoter regions - Google Patents
Applications of the transcription factor CEBP α as the transcription factor of Kiss1 promoter regions Download PDFInfo
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Abstract
The present invention discloses a kind of application of the transcription factor of transcription factor CEBP α as Kiss1 promoter regions, belongs to genetic and cell engineering technical field.The present invention is using transcription factor CEBP α as research object, use the transcription factor CEBP α of the potential combination of Bioinformatics Prediction Kiss1 promoter regions, transcription factor CEBP α are studied in gonad granulocyte on the active influence of Kiss1 promoter regions, to reach expression regulation research of the gene in gonad granulocyte.The present invention confirm for the first time by pig ovary granular cell transcription factor CEBP α the expression regulation of Kiss1 genes and the function in gonad granulocyte are studied.This research experiment is abundant in content, as a result complete, accurate.
Description
Technical field
The invention belongs to genetic and cell engineering technical fields, and in particular in sow folliculus ovarii growth course,
Applications of the transcription factor CEBP α as the transcription factor of Kiss1 promoter regions.
Background technology
The state of growing of folliculus ovarii directly determines that the height of sow reproductive performance, granular cell function effect
The growth and locking of ovarian follicle influence granular cell the study found that Kiss1 can promote the development of ovarian follicle in ovary tissue
Function.
Ovary is the reproductive organs of jenny, can provide sex hormone for jenny, and field is provided for the maturation of ovum
Institute promotes folliculus ovarii and ovum female studies have shown that the FSH and LH of hypothalamic pituitary gonadal axis secretion can act on ovary
The development of cell, FSH can promote the growth of ovary, increase the quality of ovary, and LH can promote the hair of ovarian follicle and egg mother cell
It educates, ovulation rate；The two can also promote ovarian secretion progesterone and estradiol, to promote ovarian cycle property to ovulate, by negative anti-
Feedback adjusts the release for inhibiting GnRH, FSH and LH, makes GnRH, FSH and LH that week be presented with horizontal change of ovary inner estrogen
The variation of phase property, to maintain and adjust reproductive development.And in the growth course of folliculus ovarii, flat ovarian granulosa is thin
Born of the same parents can become gradual cube, indicate that primordial follicle germinates as primary follicle, with the number of plies of gonad granulocyte
Increase, granular cell starts to break up, these variations are in the suspend mode and activation of primordial follicle, the maturation of ovum, the locking of ovarian follicle, ovum
It all plays an important role during growth of mother cell etc..The study found that the allelic mutation of Kiss1, the ovarian follicle of rat
Quantity significantly reduces, and folliculus ovarii development slows down, late period folliculus ovarii is developed, with the increase of estrogen, in pig and sheep
Hypothalamus detect that the mRNA of Kiss1 is consequently increased, but the increase earlier than GnRH, related mouse studies have shown that
Kisspeptin may directly act on ovary, participate in the formation of premature ovarian failure, the study found that the mutation of Kiss1 or missing can
To cause the mankind or mouse low promoting sexual gland hormone hypogonadism occur, it is embodied in female mice Ovarian Volume very
Small, follicular development is abnormal, and male mice testis is small, cacospermia.
CEBPs (CCAAT enhancer-binding proteins, CEBPs) is that a kind of transcription factor family CCAAT increases
Hadron binding protein can be specifically bound to the enhancer region of DNA, the proliferation of wide participation to cell, embryonic development, machine
Body energetic supersession, Apoptosis and immune response etc., by CEBP α, CEBP β, CEBP γ, CEBP δ, CEBP ε and CEBP ζ six
Member composition.When CEBPs plays a role as negative growth factor, it is necessary to there are following three functional areas, one, stable region
(SR), it is located at N-terminal, protein structure is made more to stablize；Two, active region (AD) has two regions AD1 and AD2, each other solely
It is vertical, it mutually promotes；Three, positioned at the DNA knots one of C-terminal, stable region (SR), it is located at N-terminal, protein structure is made more to stablize；Two,
There are two regions AD1 and AD2 in active region (AD), are mutually independent, mutually promote；Three, positioned at the combined areas DNA of C-terminal,
Containing bZIL protein structures, this structural domain can be closely coupled with leucine zipper region, can specificity combination DNA sequences
Row, contain acid activatable area containing the ends N-, can directly be combined with DNA, can also identify cis-acting elements.
CEBP α are to mainly passing through the expression of two ways controlling gene：The transcription for activating suppressor, in the signal of cell
Conduction, cell differentiation, cell Proliferation, stress reaction, energetic supersession and tumour generation etc. play critically important effect.It grinds
Study carefully the CEBP α genes for showing knock-out mice, the lung of mouse and the cell of liver are all unable to normal proliferative, and CEBP α mutant mices are shown
Marrow conductor increases, and the expression of mouse kidney or bone marrow B cells CEBP α or CEBP β cause the reprogramming of some B cells to be bone
Marrow family.
Invention content
In order to overcome the disadvantages and deficiencies of the prior art, the primary purpose of the present invention is that providing a kind of transcription factor CEBP
Applications of the α as the transcription factor of Kiss1 promoter regions.
Another object of the present invention is to provide the transcription factor CEBP α answering in inhibiting Kiss1 gene expressions
With.
The E2 generations in inhibiting gonad granulocyte that another object of the present invention is to provide the transcription factor CEBP α
Application.
Another object of the present invention is to provide the small interference fragments of RNA (siRNA) for inhibiting transcription factor CEBP α.
The application that it is still another object of the present invention to provide transcription factor CEBP α to influence the generation of E2 by regulating and controlling Kiss1.
The overexpression vector and RNA interfering that transcription factor CEBP α are built by technique for gene engineering, analyze the transcription because
The overexpression and interference effect of sub- CEBP α studies transcription factor CEBP α in gonad granulocyte to the shadow of the generation of E2
It rings, and then studies the application that transcription factor CEBP α influence the generation of E2 by regulating and controlling Kiss1.
The purpose of the invention is achieved by the following technical solution：
The present invention provides applications of the transcription factor CEBP α as the transcription factor of Kiss1 promoter regions.
The present invention provides applications of the transcription factor CEBP α in inhibiting Kiss1 expression.It is overexpressed transcription factor
CEBP α can inhibit the expression of Kiss1, promote the expression of Kiss1 after the expression of interference transcription factor CEBP α.
The present invention provides the transcription factor CEBP α applications that E2 is generated in inhibiting gonad granulocyte.It is overexpressed
It can inhibit the generation of E2 after transcription factor CEBP α, the generation of E2 can be promoted after the expression of interference transcription factor CEBP α.
The present invention provides the transcription factor CEBP α applications for influencing the generation of E2 by regulating and controlling Kiss1.It is overexpressed and turns
It can inhibit the facilitation that Kiss1 generates E2 after record factor CEBP α, can promote after the expression of interference transcription factor CEBP α
The facilitation that Kiss1 generates E2.
The present invention provides the siRNA for inhibiting transcription factor CEBP α, and sequence is as follows：
CEBPα-siRNA-1：5′-ACGAGACGUCCAUCGACAU-3′；
CEBPα-siRNA-2：5′-UCGACAUCAGCGCCUACAU-3′；
CEBPα-siRNA-3：5′-CCUUCAACGACGAGUUCCU-3′；
The verification result of the present invention is as follows：
1, there are transcription factor CEBP α (Gene ID for bioinformatics software prediction pig Kiss1 promoter regions:397307)
Binding site, read effects of the Literature Consult transcription factor CEBP α in folliculus ovarii growth course, primarily determine transcription because
Sub- CEBP α are the transcription factor of pig Kiss1 promoter regions.
2, ChIP separately verifies the combination situation of Kiss1 promoter regions and potential transcription factor CEBP α, the results show that
Transcription factor CEBP α can be incorporated in the region of the promoter -744 of Kiss1~-733.
3, the eukaryotic expression vector pcDNA3.1-CEBP α in the areas CDs containing transcription factor CEBP α, overexpression transcription are built
Factor CEBP α find out the concentration of most suitable transfection, are 200ng.
4, pass through Dual-Luciferase report analysis, after obtaining overexpression transcription factor CEBP α, the promoter activity of Kiss1
It reduces, is obtained in transcriptional level and translation skill verification using qRT-PCR and Western blotting：Overexpress transcription factor
After CEBP α, the mRNA and protein expression of Kiss1 are significantly reduced.
5,3 pairs of transcription factor CEBP α interference small fragment/controls (CEBP α-siRNA/Scrambled-siRNA), sieve are synthesized
It selects and detects its jamming effectiveness.Rotaring redyeing gene interferes in small fragment to gonad granulocyte, passes through qRT-PCR means, finishing screen
The preferable CEBP α-siRNA small fragments of interference effect are selected to carry out subsequent experimental.
6, transcription factor CEBP α interfere small fragment (CEBP α-siRNA) transfection particles cell, using qRT-PCR and
Western blotting are obtained in transcriptional level and translation skill verification：After the expression for interfering transcription factor CEBP α, Kiss1
MRNA and protein expression significantly increase.
7, transcription factor CEBP α are overexpressed or interfered in the gonad granulocyte of pig, and ovarian granulosa is detected with ELISA
The content of E2 in cell conditioned medium.
8, transcription factor CEBP α and Kiss1 are overexpressed or interfered in the gonad granulocyte of pig, are detected with ELISA
The content of E2 in gonad granulocyte supernatant.
The present invention uses molecule and studies it in pig with cell biology method using transcription factor CEBP α as research object
To the expression regulation of Kiss1 and function effect in gonad granulocyte.Key point is：(1) ChIP verifies transcription factor CEBP α
Kiss1 promoter regions can be combined；(2) qRT-PCR and Western blot transcriptional level and translation skill verification transcription because
Sub- CEBP α are in pig ovary granular cell to the expression regulation of Kiss1；(3) transcription factor CEBP α are overexpressed or interfere, it is right
The variation of E2 levels in sow gonad granulocyte；(4) it overexpresses or interferes transcription factor CEBP α and Kiss1, to sow ovum
The variation of E2 levels in nest granular cell.
Studies have shown that CEBP α have vital effect, Ke Nengtong in the development of rat ovary ovarian follicle and ovulation process
Cross the ovulation of EGFR-RAS-ERK accesses regulation and control mouse and the formation sheet of corpus luteum.The present invention confirms transcription factor CEBP α couple for the first time
The expression regulation of Kiss1 genes and the function in gonad granulocyte.
The present invention mechanism be：
Predict the potential Binding site for transcription factor in the gene promoter areas Kiss1, ChIP verifications in bioinformatics website
The gene promoter areas Kiss1 and potential transcription factor combination situation build the recombinant vector containing transcription factor CDs region sequences
And siRNA, transiently transfect the gonad granulocyte into pig, Dual-luciferase reportor systerm, qRT-PCR, Western Blot skills
Art analyzes influence of the transcription factor to the activity of the gene promoter areas Kiss1, mRNA level in-site and protein level.And pass through ELISA
The concentration variation of E2 in kit detection overexpression or interference transcription factor sow gonad granulocyte supernatant.
The present invention has the following advantages and effects with respect to the prior art：
First passage of the present invention in pig ovary granular cell transcription factor CEBP α to the expression regulations of Kiss1 genes and
Function in gonad granulocyte is studied.This research experiment is abundant in content, as a result complete, accurate.
Description of the drawings
Fig. 1 is ChIP verification Kiss1 promoter regions and potential transcription factor CEBP α combination situation maps；Wherein, Tu1AShi
Bioinformatics software predicts the potential Binding site for transcription factor of pig Kiss1 promoter regions；Figure 1B is that ChIP verifications Kiss1 is opened
The combination situation in mover area and potential transcription factor CEBP α.
Fig. 2 is the result figure for overexpressing transcription factor and finding out the most suitable transfection concentrations of transcription factor CEBP α.
Fig. 3 is expression regulation figures of the verification overexpression transcription factor CEBP α to Kiss1；Wherein, Fig. 3 A, Fig. 3 B, Fig. 3 C and
Fig. 3 D indicate double fluorescent reporter gene verifications, qRT-PCR and Western blot verifications respectively.
Fig. 4 is that 3 couple of qRT-PCR detection transcription factor CEBP α interferes the jamming effectiveness figure of small fragment.
Fig. 5 is expression regulation figures of the verification interference transcription factor CEBP α to Kiss1；Wherein, Fig. 5 A, Fig. 5 B and C points of Fig. 5
It Biao Shi not qRT-PCR and Western blotting verification results；CEBP α-siRNA indicate CEBP α-siRNA-2 in figure.
Fig. 6 is E2 (estradione) in gonad granulocyte supernatant after detection overexpression or interference transcription factor CEBP α
Horizontal variation diagram；Wherein, CEBP α-siRNA indicate CEBP α-siRNA-2 in figure.
Fig. 7 is that E2 is (female in gonad granulocyte supernatant after detecting overexpression or interference transcription factor CEBP α and Kiss1
Diketone) horizontal variation diagram；Wherein, pcDNA3.1-Kiss1 indicates the eukaryon table containing the areas Kiss1 gene C Ds built in figure
Up to carrier pcDNA3.1-Kiss1；Kiss1-siRNA indicates that Kiss1-siRNA-3, sequence are 5 '-
GCCACUUCCUUCAAGGAGA-3′；CEBP α-siRNA indicate CEBP α-siRNA-2；Scrambled-siRNA (K) indicates needle
To the Scrambled-siRNA of Kiss1；Scrambled-siRNA (C) indicates the Scrambled-siRNA for CEBP α.
Specific implementation mode
Present invention will now be described in further detail with reference to the embodiments and the accompanying drawings, but embodiments of the present invention are unlimited
In this.
In the following examples, the experimental methods for specific conditions are not specified, usually according to normal condition.
The culture of 1 gonad granulocyte of embodiment
(1) ovary is acquired in slaughterhouse, is placed in 37 DEG C of vacuum flask with PBS or physiological saline (dual anti-containing 1%) and is transported rapidly
Go back to laboratory；
(2) ovary of collection is transferred to rapidly after Sterile culture room cleans 3 times with preheated PBS (dual anti-containing 1%)
Superclean bench；Liquor folliculi is drawn in the shallow insertion ovary antral follicles of the sterile disposable syringes of 1mL；
(3) liquor folliculi drawn is placed in the 15mL centrifuge tubes containing appropriate DMEM, and 1000rpm room temperatures centrifuge 6min；
(4) it discards supernatant, then is resuspended with DMEM, centrifugation, repeated washing cell 2 times；Prepare DMEM complete mediums：89%
DMEM+10%FBS+1% is dual anti-；
(5) it draws cell re-suspension liquid and complete medium is inoculated in 75mL culture bottles；37 DEG C are placed in, 5%CO2In incubator
Stationary culture.
Described is dual anti-for penicillin and streptomysin.
The inoculation and transfection of 2 gonad granulocyte of embodiment
(1) granular cell is grown to 90% or so, outwell culture medium, with preheating containing it is 1% dual anti-(it is described it is dual anti-be mould
Element and streptomysin) PBS wash 3 times；
(2) trypsin digestion is added, is put into incubator 3min or so, microscopically observation to most cells levitating,
Equivalent terminate liquid is added immediately and terminates digestion；
(3) DMEM is cleaned 2 times, and period 1000rpm centrifuges 5min；
(4) cell precipitation is gently resuspended with complete medium, uniformly assigns in each hole, body is supplemented with complete medium
Product, gently shakes up, puts in incubator and cultivate；
(5) left and right, observation granular cell state prepare transfection when cell confluency degree is up to 80% or so for 24 hours；
(6) transfection method is by Invitrogen companies3000 kit specifications carry out；Often
Group 3 repetitions of setting；
(7) orifice plate after transfecting is placed in 37 DEG C, 5%CO2It is cultivated in incubator；
(8) 1~3 day after transfecting, cell state is observed, well-grown collects cell.
3 qRT-PCR of embodiment
The SYBR SYBR Green qPCR of Thermo companies of the U.S. are respectively adopted in the qRT-PCR detections of gene in the present invention
Mix kits.Using the content for comparing Ct value methods detection sample gene, specific formula for calculation is as follows for experiment：
Gene relative expression quantity=2{ < ﹙ experimental group target gene Ct Zhi ﹚-﹙ experimental group reference gene Ct Zhi ﹚ >-< ﹙ control group target gene Ct Zhi ﹚-﹙ control group reference gene Ct Zhi ﹚ > }
Internal reference wherein is done with GAPDH to genetic test, the qRT-PCR primers used in the present invention are：
qRT-PCR-Kiss1 Forward：5′-AACCAGCATCTTCTCACCAGG-3′；
Reverse：5′-CTTTCTCTCCGCACAACGC-3′；
qRT-PCR-GAPDH Forward：5′-TCCCGCCAACATCAAAT-3′；
Reverse：5′-CACGCCCATCACAAACAT-3′；
qRT-PCR-CEBPα Forward：5′-CTGAGGTCTGCCAGAAGC-3′；
Reverse：5′-AACAGAAGAAGGAAGGGAGT-3′；
For the Total RNAs extraction of cell with reference to Takara company's T RIzol operational manuals, specific extraction step is as follows：
(1) granular cell is directly added into TRIzol；
(2) 10min is placed at room temperature with abundant lytic cell, and 12000g centrifuges 5min, abandons precipitation and suct clearly in new
In RNase-free pipes；
(3) 0.2mL chloroforms are added and acutely shake 15~30s (per 1mL TRIzol), place after 5min 4 DEG C at room temperature
12000g centrifuges 15min；
(4) upper strata aqueous phase is drawn to be placed in new RNase-free EP pipes；
(5) 0.5mL isopropanols (per 1mL TRIzol) are added, 10min is being placed at room temperature for after the mixing that lightly turns upside down,
4 DEG C of 12000g centrifuge 10min；
(6) it abandons supernatant and is placed on room temperature, 75% ethyl alcohol-DEPC of 1mL (per 1mL TRIzol) are added with washing along tube wall
Supernatant is abandoned as possible after RNA, 4 DEG C of 12000g centrifugations 5min；
(7) it is dried in vacuo 5~10min, pays attention to avoiding RNA precipitate drying excessive；
(8) DEPC water is added to dissolve RNA precipitate.
The reverse transcription PCR of mRNA uses the Thermo Scientific RevertAid First of Thermo companies
Strand cDNA Synthesis Kit。
4 ELISA method of embodiment measures E2 contents in pig ovary granular cell supernatant sample
ELISA method measures E2 content detections in pig ovary granular cell supernatant sample, with reference to the Pig of CUSABIO companies
E2 ELISA kits, concrete operation step are as follows：
After (1) 24 orifice plate transfects 48h, cell culture fluid is drawn to sterile 1.5mL centrifuge tubes, -20 DEG C preserve for use.
(2) prepare sample and reagent；
(3) blank control is set；
(4) 50 μ L samples are added per hole；
(5) HRP-conjugate of 50 μ L is added per hole for sample sets, and control group is not added with, and 50 μ L antibody are then added per hole,
It is uniformly mixed；
(6) 37 DEG C of incubation 1h；
(7) it is washed three times with the Wash Buffer of 200 μ L；
(8) the Substrate B of the Substrate A and 50 μ L of 50 μ L are added per hole, are uniformly mixed, 37 DEG C of incubations
15min；
(9) the Stop Solution of 50 μ L are added per hole, gently blow and beat, are uniformly mixed, whole samples have been surveyed within 10min
The OD values of product.
5 luciferase reporter gene Activity determination of embodiment
Luciferase reporter gene Activity determination, with reference to the Dual-Luciferase Reporter of Promega companies
Assay System kits, concrete operation step are as follows：
(1) after transfecting 48h, old culture medium is sucked, is cleaned twice with PBS, the Glo of 100 μ L is added per hole cell
Lysis Buffer, room temperature slightly shake 5min, collect cell pyrolysis liquid；
(2) after 96 hole luminescent screens being added in 30 μ L cell pyrolysis liquids, 75 μ L are added wherein
Luciferase Assay Reagent stand 15~30min after mixing at 20~25 DEG C.In BioTek companies Synergy 2
Luminous value is detected on multi-function microplate reader, corresponds to the expression of firefly luciferase；
(3) 75 μ L are addedReagent reagents stand 15~30min after mixing at 20~25 DEG C.
Luminous value is detected, the expression of renilla luciferase is corresponded to；
(4) ratio of firefly luciferase and renilla luciferase expression quantity is firefly luciferase relative activity,
As correspond to target gene activity (three repetitions).
Embodiment 6ChIP
The design primer near each Binding site for transcription factor, primer sequence is as follows, is crosslinked through cell after, and ultrasound is split
Solution, chromosome immunoprecipitate, and reversed crosslinking and DNA purifying etc. carry out ChIP verifications.
Pig CEBP α gene C hIP primer sequences such as following table：
Crosslinking
(1) plus in 275 μ L, 37% formaldehyde to 10mL culture mediums (formaldehyde final concentration 1%), room temperature shaker handles 10min；
(2) plus in 500 μ 10 × glycine of L 10min is handled with unreacted formaldehyde, room temperature shaker；
(3) culture medium is thoroughly absorbed；
(4) plus 1 × PBS is pre-chilled in 1mL, and PBS is abandoned after jog washing cell；
(5) cell scraper scraping cells, enrichment is to centrifuge tube, and at 4 DEG C, 1000g centrifuges 5min, abandons supernatant；
(6) plus 1 × PBS is pre-chilled in 1mL, and after jog washs cell, at 4 DEG C, 1000g centrifuges 5min, abandons supernatant；
(7) PBS, part ultrasound cracking and -80 DEG C of preservations are exhausted.
Ultrasound cracking
(1) plus 200 μ L cell pyrolysis liquids and 1 μ L protease inhibitors, shaking table handles 30min at 4 DEG C；
(2) plus 800 μ L ChIP dilutions make final volume reach 1mL；
(3) auto-ultrasonic is crushed instrument, and amplitude 50W, 0.5s on+0.5s off handle 20min；
(4) after being crushed, 10000g centrifuges 15min at 4 DEG C, supernatant is moved in new centrifuge tube；
(5) it takes 50 μ L supernatants as input, adds 50 μ L dilutions and 5 μ L Proteinase Ks, 65 DEG C of incubation 4h.Remaining -80 DEG C
It preserves；
(6) input is purified, 50 μ L EB dissolving DNAs are finally added；
(7) 5 μ L DNA is taken to run glue verification with 2% Ago-Gel.
Chromosome immunoprecipitates
(1) the PBS washings for taking 60 μ L magnetic beads that 1mL is added to be pre-chilled；
(2) pipe is placed on magnetic frame and stands 2min, remove supernatant；
(3) plus 1 × BSA closes magnetic bead, 4 DEG C of 5min；
(4) pipe is placed on magnetic frame and stands 2min, remove supernatant；
(5) plus 1mL TE buffer solutions wash magnetic bead, and supernatant is removed after soft mixing；
(6) add 100 μ L TE buffer solutions and 3~5 μ g antibody, 4 DEG C of incubation 2h；
(7) plus in 1mL clasmatosises liquid to the pipe equipped with magnetic bead, 4 degree of overnight incubations；
Reversed crosslinking and DNA purifying
(1) plus the washing lotion I of 1mL precoolings, 4 DEG C of shaking tables handle 5min；
(2) pipe is placed on magnetic frame and stands 2min, remove supernatant；
(3) plus the washing lotion II of 1mL precoolings, 4 DEG C of shaking tables handle 5min；
(4) pipe is placed on magnetic frame and stands 2min, remove supernatant；
(5) plus the washing lotion III of 1mL precoolings, 4 DEG C of shaking tables handle 5min；
(6) pipe is placed on magnetic frame and stands 2min, remove supernatant；
(7) plus the TE buffer solutions of 1mL precoolings, 4 DEG C of shaking tables handle 5min；
(8) pipe is placed on magnetic frame and stands 2min, remove supernatant；
(9) add 200 μ L EB and 10 μ L Proteinase Ks, 65 DEG C of incubation 4h；
(10) pipe is placed on magnetic frame and stands 2min, remove supernatant；
(11) DNA after purifying crosslinking, is dissolved in the EB of 60~100 μ L, and detection or -80 DEG C of preservations are needed after progress.
Qualitative PCR
(1) reaction system (20 μ L)：
| Component | Addition (μ L) |
| ddH2O | 7 |
| 2×PCR Premix | 10 |
| Forward Prime(10pmol/μL) | 0.5 |
| Reverse Primer(10pmol/μL) | 0.5 |
| DNA | 2 |
| Total volume | 20 |
Note：Reaction solution preparation carries out on ice
(2) amplification program
Reaction condition is：95 DEG C 3 minutes, then 95 DEG C 10 seconds, 60 DEG C 30 seconds, 72 DEG C, 1 minute, totally 40 cycle, 72 DEG C 5
Minute.
(3) qualitative PCR agarose gel electrophoresis
Balance weighs in 1.6g agaroses to conical flask, pours into 80mL TBE (1 ×) buffer solution, is dissolved by heating in micro-wave oven
1min gently shakes the cooling of postposition room temperature；About 60 DEG C are cooled to, nucleic acid dye (EB substitutes) 10 μ L are added, is gently shaken mixed
It is even；Pour into be inserted with comb in offset plate, cannot have bubble；It is placed at room temperature for about 30min to solidify completely to gel, from offset plate
It takes out in gel to electrophoresis tank, it is spare.
(4) electrophoresis
Electrophoretic buffer (1 × TBE) is added in electrophoresis tank to flooding gel；Sample prepares：Take the PCR product of amplification
6 × Loding buffer, 1 μ L, mixing is added in 5 μ L；Point sample：It is gently injected in electrophoresis hole with pipette tips pipette samples, stays a hole point
Sample is 5 μ L of (100bp) Maker；Deposition condition：120V 40 minutes.
7 Western Blotting of embodiment
(1) extraction of monolayer adherence total protein of cell：Cell culture fluid is outwelled, the PBS being pre-chilled in right amount is added and washes cell three
It is secondary to wash away culture solution.Culture bottle is placed on ice after PBS is abandoned only.6 orifice plates cell per well is added 100~200 μ L albumen and splits
Solve the PMSF, lytic cell 30min of liquid and 10 μ L 100mM.It collects cell pyrolysis liquid to move in 1.5mL centrifuge tubes, 4 DEG C
14000rpm centrifuges 5min.It takes part supernatant that loading buffer is added, boils 10min.After slowly restoring room temperature, slightly centrifuges, put
In -20 DEG C of preservations；
(2) SDS-PAGE electrophoresis：After BCA methods protein sample tentatively quantifies, every group takes 20 μ g total proteins and 5 × loading buffer
Liquid presses 5:1 mixing, boils 5min.Stop SDS-PAGE electrophoresis to the rigid plastic emitting bottom of bromophenol blue；
(3) Protein transfer：Pvdf membrane methanol pre-processes 3~5s, puts to transfer liquid and infiltrates 30min.Gel is taken out, by it
It puts to filter paper, forms gel and transfer stack layer " sandwich " structure.This operation must completely remove bubble.100V constant pressures 60
~120min；
(4) immunoblotting analysis：Hybond membrane is taken out, TBST rinses 5min, in triplicate.5% skimmed milk power solution room temperature is closed
90min, TBST rinse 5min, in triplicate.PIK3R2 and TSC1 primary antibody (PIK3R2 primary antibodies are added：PIK3R2/p85 Beta,
Purchased from LSBio companies；TSC1 primary antibodies：Hamartin is purchased from biorbyt companies) (1:2000) 4 DEG C of overnight incubations, TBST wash film
5min, three times.Secondary antibody diluent (secondary antibody is added：Horseradish peroxidase-labeled goat anti-rabbit igg (H+L), it is public purchased from the green skies
Department) (1:10000) it is incubated at room temperature 1h, TBST washes film 5min, three times.Distilled water rinses film 2min, three times.It dries on pvdf membrane
Liquid is added chemiluminescent agent and is put on a film, is put into x-ray cassette and is placed in dark place.Magazine is opened in dark place, is put into glue
Piece opens magazine after 5min, takes out film.Protein band in film is analyzed using Image Plus softwares.
Interpretation of result：
1, bioinformatics software predicts the potential Binding site for transcription factor of pig Kiss1 promoter regions, and prediction result is such as
Figure 1A；Verify the combination situation of Kiss1 promoter regions and potential transcription factor CEBP α, the only primer in -744~-733 regions
The DNA fragmentation to expected size is expanded, as a result as shown in Figure 1B, it was demonstrated that transcription factor CEBP α can start in conjunction in Kiss1
Sub -744~-733 regions.
2, the carrier for expression of eukaryon containing transcription factor CEBP α CDs region sequences is built, qRT-PCR detects transcription factor
The overexpression effect of CEBP α, the results are shown in Figure 2, it was demonstrated that the overexpression significant effect of transcription factor CEBP α.
3, by Dual-Luciferase report analysis, as a result as shown in Figure 3A, after finding overexpression transcription factor CEBP α,
Kiss1 promoter activities reduce.Using qRT-PCR and Western blot transcription factor is verified in transcriptional level and translation skill
CEBP α regulation and control Kiss1 genes (Gene ID：100145896) expression, after finding overexpression transcription factor CEBP α, Kiss1's
MRNA and protein expression significantly reduce, as a result as shown in Fig. 3 B, Fig. 3 C and Fig. 3 D.
4,3 pairs of transcription factor CEBP α interference small fragments (CEBP α-siRNA) are synthesized, screens and detects its jamming effectiveness.Knot
Fruit is as shown in Figure 4.Rotaring redyeing gene interferes in small fragment to granular cell, by qRT-PCR means, finally screen interference effect compared with
Good CEBP α-siRNA small fragments carry out subsequent experimental.
CEBPα-siRNA-1：5′-ACGAGACGUCCAUCGACAU-3′；
CEBPα-siRNA-2：5′-UCGACAUCAGCGCCUACAU-3′；
CEBPα-siRNA-3：5′-CCUUCAACGACGAGUUCCU-3′；
Above-mentioned interference small fragment is synthesized by Guangzhou Ribo Bio Co., Ltd.；Scrambled-siRNA is compareed to come
From Guangzhou Ribo Bio Co., Ltd..
5, transcription factor CEBP α interfere small fragment (CEBP α-siRNA-2) transfection particles cell, research transcription factor CEBP
α interferes influence of the small fragment (CEBP α-siRNA-2) to pig Kiss1 expression regulations.Pass through qRT-PCR and Western
Blotting regulates and controls the expression of Kiss1 genes, knot in transcriptional level and translation skill verification transcription factor CEBP α interference small fragments
Fruit is as shown in Fig. 5 A, Fig. 5 B and Fig. 5 C.
6, after detecting overexpression and interference transcription factor CEBP α (CEBP α-siRNA-2) using ELISA kit, ovary
The concentration variation of E2 inhibits the generation of E2, as a result such as Fig. 6 A institutes after overexpressing transcription factor CEBP α in granulosa cell culture liquid
Show；After interfering transcription factor CEBP α, promote the generation of E2, as a result as shown in Figure 6B.
7, after detecting overexpression and interference transcription factor CEBP α using ELISA kit, after overexpressing CEBP α, can press down
Kiss1 processed secretes granular cell the facilitation of E2, as a result as shown in Figure 7 A；After the expression for interfering CEBP α, it can promote
Kiss1 secretes granular cell the facilitation of E2, and fruit is as shown in Figure 7 B.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, it is other it is any without departing from the spirit and principles of the present invention made by changes, modifications, substitutions, combinations, simplifications,
Equivalent substitute mode is should be, is included within the scope of the present invention.
Sequence table
<110>Agricultural University Of South China
<120>Applications of the transcription factor CEBP α as the transcription factor of Kiss1 promoter regions
<160> 14
<170> SIPOSequenceListing 1.0
<210> 1
<211> 19
<212> RNA
<213>Artificial sequence (Artificial Sequence)
<220>
<223> CEBPα-siRNA-1
<400> 1
acgagacguc caucgacau 19
<210> 2
<211> 19
<212> RNA
<213>Artificial sequence (Artificial Sequence)
<220>
<223> CEBPα-siRNA-2
<400> 2
ucgacaucag cgccuacau 19
<210> 3
<211> 19
<212> RNA
<213>Artificial sequence (Artificial Sequence)
<220>
<223> CEBPα-siRNA-3
<400> 3
ccuucaacga cgaguuccu 19
<210> 4
<211> 19
<212> RNA
<213>Artificial sequence (Artificial Sequence)
<220>
<223> Kiss1-siRNA-3
<400> 4
gccacuuccu ucaaggaga 19
<210> 5
<211> 21
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<220>
<223> qRT-PCR-Kiss1 Forward
<400> 5
aaccagcatc ttctcaccag g 21
<210> 6
<211> 19
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<220>
<223> qRT-PCR-Kiss1 Reverse
<400> 6
ctttctctcc gcacaacgc 19
<210> 7
<211> 17
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<220>
<223> qRT-PCR-GAPDH Forward
<400> 7
tcccgccaac atcaaat 17
<210> 8
<211> 18
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<220>
<223> qRT-PCR-GAPDH Reverse
<400> 8
cacgcccatc acaaacat 18
<210> 9
<211> 18
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<220>
<223> qRT-PCR-CEBPα Forward
<400> 9
ctgaggtctg ccagaagc 18
<210> 10
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<220>
<223> qRT-PCR-CEBPα Reverse
<400> 10
aacagaagaa ggaagggagt 20
<210> 11
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<220>
<223> ChIP-CEBPα F
<400> 11
cactgtgcaa gtggtctcgg 20
<210> 12
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<220>
<223> ChIP-CEBPα R
<400> 12
cccgttagaa tgtgccttcc 20
<210> 13
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<220>
<223> ChIP-GAPDH F
<400> 13
gatgtcctga gcccctacag 20
<210> 14
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<220>
<223> ChIP-GAPDH R
<400> 14
ggtaggtgat ggggactgag 20
Claims (7)
1. applications of the transcription factor CEBP α as the transcription factor of Kiss1 promoter regions.
2. applications of the transcription factor CEBP α in inhibiting Kiss1 expression.
3. the transcription factor CEBP α applications that E2 is generated in inhibiting gonad granulocyte.
4. the application that transcription factor CEBP α influence the generation of E2 by regulating and controlling Kiss1.
5. inhibiting the siRNA of transcription factor CEBP α, it is characterised in that：The sequence of the siRNA is as follows：CEBPα-siRNA-1：
5′-ACGAGACGUCCAUCGACAU-3′。
6. inhibiting the siRNA of transcription factor CEBP α, it is characterised in that：The sequence of the siRNA is as follows：CEBPα-siRNA-2：
5′-UCGACAUCAGCGCCUACAU-3′。
7. inhibiting the siRNA of transcription factor CEBP α, it is characterised in that：The sequence of the siRNA is as follows：CEBPα-siRNA-3：
5′-CCUUCAACGACGAGUUCCU-3′。
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| CN114349841B (en) * | 2021-10-26 | 2024-02-13 | 安徽农业大学 | Transcription factor for regulating and controlling expression activity of OVR gene on follicular membrane surface and application thereof |
| CN114874993A (en) * | 2022-05-30 | 2022-08-09 | 华南农业大学 | Method for regulating and controlling pig ovarian granulosa cell MMP2 gene expression |
| CN114874993B (en) * | 2022-05-30 | 2023-08-15 | 华南农业大学 | Method for regulating and controlling MMP2 gene expression of porcine ovarian granulosa cells |
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