CN108715848B - Application of transcription factor CEBP alpha as transcription factor of Kiss1 promoter region - Google Patents

Abstract

The invention discloses application of a transcription factor CEBP alpha as a transcription factor of a Kiss1 promoter region, belonging to the technical field of genetic engineering and cell engineering. The invention takes a transcription factor CEBP alpha as a research object, adopts the transcription factor CEBP alpha for predicting potential binding of a Kiss1 promoter region by bioinformatics, and researches the influence of the transcription factor CEBP alpha on the activity of a Kiss1 promoter region in ovarian granulosa cells so as to achieve the expression regulation research of the gene in the ovarian granulosa cells. The invention proves that the expression regulation of Kiss1 gene and the function of the gene in the ovary granular cells are researched by the transcription factor CEBP alpha in the pig ovary granular cells for the first time. The research experiment has rich content, complete and accurate results.

Description

Application of transcription factor CEBP alpha as transcription factor of Kiss1 promoter region

Technical Field

The invention belongs to the technical field of genetic engineering and cell engineering, and particularly relates to application of a transcription factor CEBP alpha as a transcription factor of a Kiss1 promoter region in a sow ovarian follicle development process.

Background

The growth and development state of ovarian follicles directly determines the reproductive performance of sows, and the granulosa cell function influences the growth and atresia of the follicles, so that the research shows that the Kiss1 can promote the development of the follicles in the ovarian tissues and influence the function of the granulosa cells.

The ovary is a reproductive organ of a female animal, can provide sex hormone for the female animal and provides a place for the maturation of the ovum, and researches show that FSH and LH secreted by a hypothalamus-pituitary-gonad axis can act on the ovary to promote the development of ovarian follicles and oocytes, the FSH can promote the growth of the ovary to increase the quality of the ovary, and the LH can promote the development of the ovarian follicles and the oocytes to promote ovulation; the two can also promote the ovary to secrete progesterone and estradiol, so that the ovary can be promoted to periodically ovulate, and the release of GnRH, FSH and LH is inhibited through negative feedback regulation, so that the GnRH, FSH and LH show periodic change along with the change of the level of the estrogen in the ovary, and the reproductive development is maintained and regulated. During the development of ovarian follicles, flat ovarian granulosa cells become gradually cuboidal, which indicates that primordial follicles begin to develop into primary follicles, and as the number of layers of ovarian granulosa cells increases, granulosa cells begin to differentiate, and these changes play an important role in the dormancy and activation of primordial follicles, maturation of ova, atresia of follicles, growth of oocytes, and the like. Researches show that the allelic gene of Kiss1 is mutated, the number of follicles in rats is obviously reduced, the ovarian follicle development is slowed down, mRNA of Kiss1 is detected to be increased in hypothalamus of pigs and sheep along with the increase of estrogen at the late stage of the ovarian follicle development, but the increase is earlier than that of GnRH, and researches on mice show that kisspeptin can directly act on ovaries and participate in the formation of premature ovarian failure, and researches show that mutation or deletion of Kiss1 can cause the hypogonadotropic gonadal function of human or mice to appear, and the hypogonadotropic gonadal function is particularly shown in that female mice have small ovarian size, abnormal follicle development, small testes of male mice and poor sperm development.

CEBPs (CCAAT enhancer-binding proteins, CEBPs) are a class of transcription factor family CCAAT enhancer binding proteins, can specifically bind to the enhancer region of DNA, and are widely involved in cell proliferation, embryonic development, energy metabolism, apoptosis, immune response, etc., and are composed of six members, CEBP α, CEBP β, CEBP γ, CEBP δ, CEBP ε, and CEBP ζ. When CEBPs function as negative regulators, three functional regions, namely a Stabilizing Region (SR) and a Stable Region (SR), must exist at the N terminal, so that the protein structure is more stable; an activation region (AD) which comprises two regions AD1 and AD2, wherein the two regions are independent of each other and promote each other; thirdly, a DNA knot I and a Stable Region (SR) which are positioned at the C terminal and positioned at the N terminal enable the protein structure to be more stable; an activation region (AD) which comprises two regions AD1 and AD2, wherein the two regions are independent of each other and promote each other; and the DNA binding region positioned at the C terminal contains a bZIL protein structure, the domain can be closely connected with the leucine zipper region, can be specifically bound with a DNA sequence, contains an acidic activation region at the N terminal, can be directly bound with the DNA, and can also recognize a cis-acting element.

CEBP α pairs regulate gene expression primarily in two ways: activation of transcription of suppressor genes plays an important role in cell signaling, cell differentiation, cell proliferation, stress response, energy metabolism, tumorigenesis, and the like. Studies have shown that knockout of the CEBP α gene in mice, and failure of cells in the lungs and liver of mice to proliferate normally, CEBP α mutant mice show increased bone marrow leads, and expression of CEBP α or CEBP β in mouse kidney or bone marrow B cells results in reprogramming of some B cells into bone marrow families.

Disclosure of Invention

In order to overcome the defects and shortcomings of the prior art, the primary object of the present invention is to provide an application of the transcription factor CEBP alpha as a transcription factor of Kiss1 promoter region.

The invention also aims to provide the application of the transcription factor CEBP alpha in inhibiting the expression of Kiss1 gene.

The invention also aims to provide the application of the transcription factor CEBP alpha in inhibiting the generation of E2 in ovarian granulosa cells.

Another objective of the invention is to provide RNA small interference fragments (siRNA) for inhibiting the transcription factor CEBP alpha.

It is still another object of the present invention to provide the use of the transcription factor CEBP α to influence the production of E2 by modulating Kiss 1.

The overexpression vector and the interference RNA of the transcription factor CEBP alpha are constructed by a genetic engineering technology, the overexpression and the interference effect of the transcription factor CEBP alpha are analyzed, the influence of the transcription factor CEBP alpha on the generation of E2 in ovarian granulosa cells is researched, and the application of the transcription factor CEBP alpha in influencing the generation of E2 by regulating Kiss1 is further researched.

The purpose of the invention is realized by the following technical scheme:

the invention provides an application of a transcription factor CEBP alpha as a transcription factor of a Kiss1 promoter region.

The invention provides application of the transcription factor CEBP alpha in inhibiting expression of Kiss 1. Overexpression of the transcription factor CEBP alpha can inhibit the expression of Kiss1, and interference with the expression of the transcription factor CEBP alpha can promote the expression of Kiss 1.

The invention provides application of the transcription factor CEBP alpha in inhibiting the generation of E2 in ovarian granulosa cells. The production of E2 can be inhibited after the transcription factor CEBP alpha is over-expressed, and the production of E2 can be promoted after the expression of the transcription factor CEBP alpha is interfered.

The invention provides application of the transcription factor CEBP alpha in influencing the generation of E2 by regulating Kiss 1. After the transcription factor CEBP alpha is over-expressed, the promotion effect of Kiss1 on the generation of E2 can be inhibited, and after the expression of the transcription factor CEBP alpha is interfered, the promotion effect of Kiss1 on the generation of E2 can be promoted.

The invention provides siRNA for inhibiting transcription factor CEBP alpha, which has the following sequence:

CEBPα-siRNA-1：5′-ACGAGACGUCCAUCGACAU-3′；

CEBPα-siRNA-2：5′-UCGACAUCAGCGCCUACAU-3′；

CEBPα-siRNA-3：5′-CCUUCAACGACGAGUUCCU-3′；

the verification results of the invention are as follows:

1. bioinformatics software predicts that the porcine Kiss1 promoter region has a binding site of the transcription factor CEBP alpha (Gene ID:397307), reads the literature and consults the role of the transcription factor CEBP alpha in the ovarian follicle development process, and preliminarily determines that the transcription factor CEBP alpha is the transcription factor of the porcine Kiss1 promoter region.

2. ChIP verifies the binding between the Kiss1 promoter region and the potential transcription factor CEBP alpha respectively, and the result shows that the transcription factor CEBP alpha can be bound in the promoter-744 to-733 regions of Kiss 1.

3. Constructing eukaryotic expression vector pcDNA3.1-CEBP alpha containing CDs region of transcription factor CEBP alpha, and over-expressing transcription factor CEBP alpha to find out the optimum transfection concentration of 200 ng.

4. After the over-expression transcription factor CEBP alpha is obtained through dual-luciferase report analysis, the activity of the promoter of the Kiss1 is reduced, and the expression transcription factor CEBP alpha is obtained through verification of the transcription level and the translation level by adopting qRT-PCR and Western blotting: expression of Kiss1 at mRNA and protein levels was significantly reduced after overexpression of the transcription factor CEBP α.

5. 3 pairs of transcription factor CEBP alpha interfering small fragments/controls (CEBP alpha-siRNA/Scambrelled-siRNA) were synthesized, screened and tested for their interference efficiency. Transfecting the gene interference small fragment into an ovary granular cell, and finally screening the CEBP alpha-siRNA small fragment with better interference effect by a qRT-PCR method for subsequent experiments.

6. The transcription factor CEBP alpha interference small fragment (CEBP alpha-siRNA) transfects granular cells, and the results are verified at the transcription level and the translation level by adopting qRT-PCR and Western blotting: expression of Kiss1 at mRNA and protein levels was significantly elevated after interfering with expression of the transcription factor CEBP α.

7. The transcription factor CEBP alpha is over-expressed or interfered in the ovary granular cells of pigs, and the content of E2 in the supernatant of the ovary granular cells is detected by ELISA.

8. The transcription factors CEBP alpha and Kiss1 were overexpressed or interfered with in porcine ovarian granulosa cells, and the content of E2 in the supernatant of ovarian granulosa cells was measured by ELISA.

The invention takes the transcription factor CEBP alpha as a research object, and adopts a molecular and cell biological method to research the expression regulation and function influence of the transcription factor CEBP alpha on Kiss1 in the porcine ovarian granulosa cells. The key points are as follows: (1) ChIP verifies that the transcription factor CEBP alpha can be combined with the Kiss1 promoter region; (2) the expression regulation and control of a transcription factor CEBP alpha on Kiss1 in the porcine ovarian granulosa cells are verified at the transcription level and the translation level by qRT-PCR and Western blot; (3) overexpression or interference of transcription factor CEBP alpha, change of E2 level in sow ovarian granulosa cells; (4) overexpression or interference with transcription factors CEBP α and Kiss1, changes in E2 levels in sow ovarian granulosa cells.

Research shows that CEBP alpha plays a vital role in the development and ovulation process of ovarian follicles of rats and can regulate the ovulation and the formation of corpus luteum of mice through an EGFR-RAS-ERK pathway. The invention proves the expression regulation and the function of the transcription factor CEBP alpha on Kiss1 gene in ovarian granulosa cells for the first time.

The mechanism of the invention is as follows:

the bioinformatics website predicts potential transcription factor binding sites of the Kiss1 gene promoter region, ChIP verifies the binding condition of the Kiss1 gene promoter region and the potential transcription factors, constructs a recombinant vector and siRNA containing a transcription factor CDs region sequence, transiently transfects the recombinant vector and siRNA into the ovary granular cells of pigs, and analyzes the influence of the transcription factors on the activity, mRNA level and protein level of the Kiss1 gene promoter region by using a dual-luciferase reporter system, qRT-PCR and Western Blot technology. And detecting the concentration change of E2 in the supernatant of the ovarian granulosa cells of the sow, which are over-expressed or interfere with the transcription factors, by an ELISA kit.

Compared with the prior art, the invention has the following advantages and effects:

the invention researches the expression regulation of Kiss1 gene and the function of the gene in the ovary granular cells through a transcription factor CEBP alpha in the pig ovary granular cells for the first time. The research experiment has rich content, complete and accurate results.

Drawings

FIG. 1 is a graph of ChIP verifying the binding of Kiss1 promoter region to the potential transcription factor CEBP α; wherein, FIG. 1A shows the prediction of potential transcription factor binding sites of the promoter region of the porcine Kiss1 by bioinformatics software; FIG. 1B is a ChIP demonstration of the binding of Kiss1 promoter region to the potential transcription factor CEBP α.

FIG. 2 is a graph showing the results of finding the optimum transfection concentration of the transcription factor CEBP alpha by overexpressing the transcription factor.

FIG. 3 is a graph showing the expression control of Kiss1 by the over-expressed transcription factor CEBP α; wherein, FIG. 3A, FIG. 3B, FIG. 3C and FIG. 3D respectively show dual fluorescence reporter gene validation, qRT-PCR and Western blot validation.

FIG. 4 is a graph of interference efficiency of qRT-PCR detection of 3 pairs of interfering small fragments of the transcription factor CEBP alpha.

FIG. 5 is a graph showing the regulation of expression of Kiss1 by interfering transcription factor CEBP α; wherein, FIG. 5A, FIG. 5B and FIG. 5C respectively show the results of qRT-PCR and Western blotting; CEBP α -siRNA in the figure represents CEBP α -siRNA-2.

FIG. 6 is a graph showing the change in the level of E2 (estradiol) in the supernatant of ovarian granulosa cells following detection of overexpression or interference with the transcription factor CEBP α; wherein CEBP α -siRNA in the figure represents CEBP α -siRNA-2.

FIG. 7 is a graph showing the change in E2 (estradiol) levels in the supernatant of ovarian granulosa cells following detection of overexpression or interference of the transcription factors CEBP α and Kiss 1; wherein pcDNA3.1-Kiss1 shows the constructed eukaryotic expression vector pcDNA3.1-Kiss1 containing the CDs region of the Kiss1 gene; the Kiss1-siRNA represents Kiss1-siRNA-3 with the sequence of 5'-GCCACUUCCUUCAAGGAGA-3'; CEBP alpha-siRNA represents CEBP alpha-siRNA-2; Scrambled-siRNA (K) represents Scrambled-siRNA against Kiss 1; (C) represents a Scambled-siRNA against CEBP α.

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.

The experimental methods in the following examples, which are not specified under specific conditions, are generally performed under conventional conditions.

EXAMPLE 1 culture of ovarian granulosa cells

(1) Collecting ovaries in a slaughterhouse, placing the ovaries in PBS or normal saline (containing 1% double antibody) in a vacuum flask at 37 ℃, and quickly transporting the ovaries back to a laboratory;

(2) cleaning the collected ovaries for 3 times in a sterile culture room by using preheated PBS (containing 1% double antibody), and quickly transferring the ovaries to a superclean bench; the ovarian follicle fluid is absorbed by a 1mL sterile disposable syringe which is inserted into the ovarian follicle with a cavity in a shallow way;

(3) placing the follicular fluid into a 15mL centrifuge tube containing a proper amount of DMEM, and centrifuging at 1000rpm at room temperature for 6 min;

(4) discarding the supernatant, then resuspending and centrifuging by DMEM, and repeatedly cleaning the cells for 2 times; preparing a DMEM complete culture medium: 89% DMEM + 10% FBS + 1% double antibody;

(5) sucking the cell resuspension and complete culture medium and inoculating the cell resuspension and complete culture medium in a 75mL culture bottle; standing at 37 deg.C for 5% CO₂And (5) standing and culturing in an incubator.

The double-resistant is penicillin and streptomycin.

Example 2 inoculation and transfection of ovarian granulosa cells

(1) Growing the granular cells to about 90%, pouring out the culture medium, and washing for 3 times by using preheated PBS containing 1% double antibodies (the double antibodies are penicillin and streptomycin);

(2) adding trypsin for digestion, placing in an incubator for about 3min, observing under a microscope until most cells float, and immediately adding equivalent stop solution to stop digestion;

(3) washing with DMEM for 2 times, and centrifuging at 1000rpm for 5 min;

(4) lightly resuspending the cell sediment with complete culture medium, uniformly distributing the cell sediment into each hole, supplementing the volume with complete culture medium, lightly shaking up, and culturing in an incubator;

(5) observing the state of the granular cells after about 24 hours, and preparing transfection when the confluence degree of the cells reaches about 80%;

(6) transfection method Invitrogen

3000 kit instructions; each set was set to 3 replicates;

(7) the transfected well plates were placed at 37 ℃ in 5% CO₂Culturing in an incubator;

(8) and (5) observing the cell state 1-3 days after transfection, and collecting the cells after the cells grow well.

Example 3 qRT-PCR

The qRT-PCR detection of the gene in the invention adopts SYBR SYBR Green qPCR Mix kit of the U.S. Thermo company respectively. The content of the sample gene is detected by adopting a Ct value comparison method in the experiment, and the specific calculation formula is as follows:

relative gene expression level of 2^{- { (lead) desired gene Ct value in experimental group-lead (reference gene Ct value in experimental group) < - > (lead) desired gene Ct value in control group- < - >}

Wherein GAPDH is used as an internal reference for gene detection, and qRT-PCR primers used by the invention are as follows:

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′；

total RNA extraction of cells was performed according to the instructions of TRIzol of Takara, and the specific extraction procedure was as follows:

(1) adding the granular cells into TRIzol directly;

(2) standing at room temperature for 10min to fully lyse cells, centrifuging at 12000g for 5min, discarding the precipitate, and taking the supernatant in a new RNase-free tube;

(3) adding 0.2mL of chloroform (1 mL of TRIzol) and shaking vigorously for 15-30 s, standing at room temperature for 5min, and centrifuging at 4 ℃ and 12000g for 15 min;

(4) absorbing the upper aqueous phase and placing the upper aqueous phase in a new RNase-free EP tube;

(5) adding 0.5mL of isopropanol (per 1mL of TRIzol), gently inverting and mixing, standing at room temperature for 10min, and centrifuging at 4 ℃ at 12000g for 10 min;

(6) discarding the supernatant, placing at room temperature, adding 1mL 75% ethanol-DEPC (per 1mL TRIzol) along the tube wall to wash RNA, centrifuging at 4 ℃ at 12000g for 5min, and discarding the supernatant as much as possible;

(7) vacuum drying for 5-10 min, and taking care to avoid excessive drying of RNA precipitate;

(8) DEPC water was added to dissolve the RNA pellet.

Reverse transcription PCR of mRNA was performed using the Thermo Scientific RevertAID First Strand cDNA Synthesis Kit from Thermo corporation.

Example 4 determination of E2 content in porcine ovarian granulosa cell supernatant samples by ELISA

The ELISA method for detecting the content of E2 in the porcine ovarian granulosa cell supernatant sample refers to the Pig E2 ELISA kit of CUSABIO company, and comprises the following specific operation steps:

(1) after 48h of transfection in 24-well plates, the cell culture fluid was aspirated into sterile 1.5mL centrifuge tubes and stored at-20 ℃ until use.

(2) Preparing a sample and a reagent;

(3) setting a blank contrast;

(4) add 50. mu.L of sample per well;

(5) adding 50 μ L of HRP-conjugate to each well of the sample group, adding no HRP-conjugate to the control group, adding 50 μ L of antibody to each well, and mixing well;

(6) incubating at 37 ℃ for 1 h;

(7) washed three times with 200. mu.L of Wash Buffer;

(8) add 50. mu.L of Substrate A and 50. mu.L of Substrate B to each well, mix well, incubate for 15min at 37 ℃;

(9) add 50 μ L of Stop Solution into each well, blow gently, mix well, measure the OD value of the complete sample within 10 min.

Example 5 luciferase reporter Activity assays

The Luciferase Reporter gene activity detection refers to a Dual-Luciferase Reporter Assay System kit of Promega company, and the specific operation steps are as follows:

(1) after transfection for 48h, the old medium was aspirated off, washed twice with PBS, 100. mu.L of Glo Lysis Buffer was added to each well of cells, shaken slightly at room temperature for 5min, and cell lysates were collected;

(2) after 30. mu.L of cell lysate was added to a 96-well plate, 75. mu.L of the lysate was added thereto

And (3) uniformly mixing the Luciferase Assay Reagent, and standing for 15-30 min at 20-25 ℃. Detecting a luminous value on a multifunctional microplate reader of Synergy 2 of BioTek company, wherein the luminous value corresponds to the expression level of the firefly luciferase;

(3) then 75. mu.L of the solution was added

And (3) uniformly mixing the Reagent, and standing for 15-30 min at 20-25 ℃. Detecting a luminescence value corresponding to the level of renilla luciferase expression;

(4) the ratio of the expression amounts of the firefly luciferase and the Renilla luciferase is the relative activity of the firefly luciferase, which is the activity of the corresponding target gene (three repeats).

Example 6ChIP

Primers were designed near each transcription factor binding site, with the following sequences, followed by ChIP validation via cell cross-linking, sonication, chromosome immunoprecipitation, reverse cross-linking, and DNA purification.

The ChIP primer sequences of the pig CEBP alpha gene are shown in the following table:

cross-linking

(1) Adding 275 μ L of 37% formaldehyde into 10mL of the culture medium (formaldehyde concentration 1%), and shaking at room temperature for 10 min;

(2) adding 500 μ L10 × glycine to neutralize unreacted formaldehyde, and shaking at room temperature for 10 min;

(3) completely absorbing and removing the culture medium;

(4) adding 1mL of precooled 1 XPBS, washing the cells by gentle shaking, and then discarding the PBS;

(5) scraping cells, enriching the cells into a centrifuge tube, centrifuging the centrifuge tube at the temperature of 4 ℃ for 5min at 1000g, and removing supernatant;

(6) adding 1mL of precooled 1 XPBS, washing cells with light shaking, centrifuging for 5min at the temperature of 4 ℃ at 1000g, and removing supernatant;

(7) PBS was aspirated, partially sonicated and stored at-80 ℃.

Ultrasonic pyrolysis

(1) Adding 200 μ L cell lysate and 1 μ L protease inhibitor, and treating with shaking table at 4 deg.C for 30 min;

(2) add 800. mu.L ChIP diluent to reach 1mL final volume;

(3) an automatic ultrasonic crusher with an amplitude of 50W for 20min at 0.5s on +0.5s off;

(4) after crushing, centrifuging at 4 ℃ for 15min at 10000g, and transferring the supernatant into a new centrifuge tube;

(5) 50. mu.L of the supernatant was taken as input, 50. mu.L of the dilution and 5. mu.L of proteinase K were added, and incubation was carried out at 65 ℃ for 4 hours. Storing the rest at-80 deg.C;

(6) purifying input, and finally adding 50 mu L of EB to dissolve DNA;

(7) 5 μ L of DNA was run on a 2% agarose gel for validation.

Chromosome immunoprecipitation

(1) 60 mu L of magnetic beads are added with 1mL of precooled PBS for washing;

(2) placing the tube on a magnetic frame, standing for 2min, and removing the supernatant;

(3) adding 1 XBSA to block the magnetic beads, and keeping the temperature at 4 ℃ for 5 min;

(4) placing the tube on a magnetic frame, standing for 2min, and removing the supernatant;

(5) adding 1mL of TE buffer solution to wash the magnetic beads, gently mixing uniformly, and removing supernatant;

(6) adding 100 mu L of TE buffer solution and 3-5 mu g of antibody, and incubating for 2h at 4 ℃;

(7) adding 1mL of cell disruption solution into a tube filled with magnetic beads, and incubating overnight at 4 ℃;

reverse crosslinking and DNA purification

(1) Adding 1mL of precooled washing liquor I, and carrying out shaking table treatment at 4 ℃ for 5 min;

(2) placing the tube on a magnetic frame, standing for 2min, and removing the supernatant;

(3) adding 1mL of precooled washing liquid II, and carrying out shaking table treatment at 4 ℃ for 5 min;

(4) placing the tube on a magnetic frame, standing for 2min, and removing the supernatant;

(5) adding 1mL of precooled lotion III, and carrying out shaking table treatment at 4 ℃ for 5 min;

(6) placing the tube on a magnetic frame, standing for 2min, and removing the supernatant;

(7) adding 1mL of precooled TE buffer solution, and carrying out shaking table treatment at 4 ℃ for 5 min;

(8) placing the tube on a magnetic frame, standing for 2min, and removing the supernatant;

(9) adding 200 mu L EB and 10 mu L proteinase K, and incubating for 4h at 65 ℃;

(10) placing the tube on a magnetic frame, standing for 2min, and removing the supernatant;

(11) and purifying the crosslinked DNA, dissolving the DNA in 60-100 mu L of EB, and carrying out detection or storing at-80 ℃.

Qualitative PCR

(1) Reaction system (20 μ L):

components	Addition amount (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: the reaction solution was prepared on ice

(2) Amplification procedure

The reaction conditions are as follows: 3 minutes at 95 ℃ followed by 10 seconds at 95 ℃, 30 seconds at 60 ℃, 1 minute at 72 ℃ for a total of 40 cycles at 72 ℃ for 5 minutes.

(3) Qualitative PCR agarose gel electrophoresis

Weighing 1.6g of agarose into a conical flask by a balance, pouring 80mL of TBE (1 x) buffer solution, heating and dissolving for 1min in a microwave oven, slightly shaking, and cooling at room temperature; cooling to about 60 deg.C, adding 10 μ L nucleic acid dye (EB substitute), gently shaking and mixing; pouring the mixture into a rubber plate inserted with a comb, wherein air bubbles cannot exist; standing at room temperature for about 30min until the gel is completely solidified, and taking out the gel from the gel preparation plate to an electrophoresis tank for later use.

(4) Electrophoresis

Adding electrophoresis buffer (1 × TBE) to the electrophoresis tank until the gel is submerged; sample preparation: adding 5 mu L of amplified PCR product into 1 mu L of 6 XLoding buffer, and mixing uniformly; sample application: sucking the sample by a gun head, gently injecting the sample into the electrophoresis hole, and reserving a hole and spotting the sample as 5 mu L of (100bp) marker; electrophoresis conditions: 120V for 40 minutes.

Example 7 Western Blotting

(1) Extraction of total protein from adherent monolayer cells: the cell culture was decanted and the cells were washed three times with an appropriate amount of pre-cooled PBS to wash out the culture. After discarding the PBS, the flasks were placed on ice. And adding 100-200 mu L of protein lysate and 10 mu L of 100mM PMSF into each well of 6-well plate cells, and lysing the cells for 30 min. The cell lysate was collected and transferred to a 1.5mL centrifuge tube and centrifuged at 14000rpm at 4 ℃ for 5 min. And adding part of the supernatant into the sample buffer, and boiling for 10 min. Slowly recovering to room temperature, centrifuging, and storing at-20 deg.C;

(2) SDS-PAGE electrophoresis: after initial quantification of protein samples by BCA method, 20. mu.g of total protein per group was mixed with 5 Xloading buffer at a ratio of 5:1 and boiled for 5 min. Performing SDS-PAGE electrophoresis until the bromophenol blue just comes out of the gel bottom;

(3) protein transfer: and (3) pretreating the PVDF membrane for 3-5 s by using methanol, and soaking the PVDF membrane in a transfer printing liquid for 30 min. Taking out the gel, and placing the gel on filter paper to form a sandwich structure of a gel transfer printing accumulation layer. This operation must remove the bubbles completely. Constant pressure of 100V for 60-120 min;

(4) immunoblotting: the hybridization membrane was removed, rinsed for 5min in TBST and repeated three times. 5% skimmed milk powder solution was blocked at room temperature for 90min, and rinsed with TBST for 5min, repeated three times. PIK3R2 and TSC1 primary antibodies (PIK3R2 primary antibody: PIK3R2/p85 Beta, available from LSBio Inc.; TSC1 primary antibody: Hamatin, available from biorbyt Inc.) (1:2000) were added and incubated overnight at 4 ℃ and membranes washed with TBST for 5min three times. Adding a second antibody diluent (a second antibody: horseradish peroxidase-labeled goat anti-rabbit IgG (H + L) purchased from Biyuntian corporation (1:10000), incubating at room temperature for 1H, washing the membrane with TBST for 5min, and carrying out three times. The membrane was rinsed with distilled water for 2min, three times. Wiping off the liquid on the PVDF film, adding a chemiluminescent agent, placing on a film, placing in an X-ray cassette, and placing in a dark room. Opening the film box in the darkroom, putting in the film, opening the film box after 5min, and taking out the film. Protein bands in the films were analyzed using Image Plus software.

And (4) analyzing results:

1. the bioinformatics software predicts the potential transcription factor binding site of the promoter region of the pig Kiss1, and the prediction result is shown in FIG. 1A; when the binding between the Kiss1 promoter region and the potential transcription factor CEBP alpha is verified, only primers of the-744 to-733 region are amplified to a DNA fragment with an expected size, and the result is shown in FIG. 1B, which proves that the transcription factor CEBP alpha can be bound to the-744 to-733 region of the Kiss1 promoter.

2. The eukaryotic expression vector containing the sequence of the CEBP alpha CDs region of the transcription factor is constructed, qRT-PCR is used for detecting the over-expression effect of the CEBP alpha of the transcription factor, and the result is shown in figure 2, which proves that the over-expression effect of the CEBP alpha of the transcription factor is obvious.

3. As a result of the dual-luciferase reporter assay, as shown in FIG. 3A, it was found that Kiss1 promoter activity was decreased after overexpression of the transcription factor CEBP α. The expression of Kiss1 Gene (Gene ID: 100145896) regulated by the transcription factor CEBP alpha was verified at the transcription level and the translation level by using qRT-PCR and Western blot, and the expression of mRNA and protein levels of Kiss1 was significantly reduced after the transcription factor CEBP alpha was overexpressed, and the results are shown in FIG. 3B, FIG. 3C and FIG. 3D.

4. 3 pairs of transcription factor CEBP alpha interference small fragments (CEBP alpha-siRNA) are synthesized, screened and tested for interference efficiency. The results are shown in FIG. 4. Transfecting the gene interference small fragment into a particle cell, and finally screening the CEBP alpha-siRNA small fragment with better interference effect by a qRT-PCR means for subsequent experiments.

CEBPα-siRNA-1：5′-ACGAGACGUCCAUCGACAU-3′；

CEBPα-siRNA-2：5′-UCGACAUCAGCGCCUACAU-3′；

CEBPα-siRNA-3：5′-CCUUCAACGACGAGUUCCU-3′；

The small interference fragments are synthesized by Ribo Biotech, Inc., Guangzhou; control Scarambled-siRNA was from Ruibo Biotech, Inc., Guangzhou.

5. The transcription factor CEBP alpha interference small fragment (CEBP alpha-siRNA-2) transfects granular cells, and the influence of the transcription factor CEBP alpha interference small fragment (CEBP alpha-siRNA-2) on the regulation and control of porcine Kiss1 expression is studied. The interference of the transcription factor CEBP alpha with the small fragment regulation of the expression of the Kiss1 gene was verified at the transcription level and the translation level by qRT-PCR and Western blotting, and the results are shown in FIG. 5A, FIG. 5B and FIG. 5C.

6. After detecting the overexpression and the interference transcription factor CEBP alpha (CEBP alpha-siRNA-2) by using an ELISA kit, the concentration of E2 in the culture solution of the ovarian granulosa cells changes, and after the overexpression of the transcription factor CEBP alpha, the generation of E2 is inhibited, and the result is shown in FIG. 6A; interfering with the transcription factor CEBP α promotes the production of E2, the results are shown in fig. 6B.

7. After detecting the over-expression and interference transcription factor CEBP alpha by using an ELISA kit, the promotion effect of Kiss1 on E2 secretion of granulosa cells can be inhibited after the over-expression of CEBP alpha, and the result is shown in FIG. 7A; interference with CEBP α expression may promote the effect of Kiss1 in promoting E2 secretion by granulosa cells, as shown in fig. 7B.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Sequence listing

<110> southern China university of agriculture

<120> use of transcription factor CEBP alpha as transcription factor of Kiss1 promoter region

<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 (4)

1. Transcription factor CEBP alpha in inhibiting swine ovarian granular cellsKiss1Use of an expression, characterized in that:

transcription factor CEBP alpha is combined in swine ovary granular cellsKiss1The promoter-744 to-733 region to repress the expression.

2. Transcription factor CEBP alpha by inhibiting in pig ovary granular cellKiss1Use of an expression to inhibit the production of E2, wherein:

transcription factor CEBP alpha is combined in swine ovary granular cellsKiss1The promoter-744 to-733 region to repress the expression.

3. siRNA that inhibits transcription factor CEBP α, characterized by: the sequences of the siRNAs are as follows:

CEBPα-siRNA-2：5′-UCGACAUCAGCGCCUACAU-3′。

4. siRNA that inhibits transcription factor CEBP α, characterized by: the sequences of the siRNAs are as follows:

CEBPα-siRNA-3：5′-CCUUCAACGACGAGUUCCU-3′。

Images