CN110106182B - Application of p65 gene in porcine ovarian granulosa cells - Google Patents

Abstract

The invention discloses application of a p65 gene in porcine ovarian granulosa cells. The invention detects the relative expression quantity of p65 mRNA in immature and mature ovarian tissues of pigs and follicular granulosa cells with different sizes, detects the proliferation, apoptosis and estrogen secretion conditions of the granulosa cells after over-expression and p65 interference, and is a molecular mechanism accumulation material in the development process of ovarian follicles of sows. Meanwhile, the results of the invention show that the p65 gene participates in promoting the proliferation and the estrogen secretion of granulosa cells, activates an estrogen signal channel, and regulates the expression of related genes ESR1, ESR2, HSD3B1, HSD17B4 and CYP 19A.

Description

Application of p65 gene in porcine ovarian granulosa cells

Technical Field

The invention belongs to the technical field of cell engineering and genetic engineering, and particularly relates to application of a p65 gene in porcine ovarian granulosa cells.

Background

Granulosa cells, which are an important component of follicles, are the main sites of estrogen synthesis, and gradually proliferate from a flat monolayer to a columnar multilayer during follicular development, and produce cytokines, growth factors, estrogens and the like associated with follicular development, thereby promoting follicular maturation. It is currently believed that the process of follicle recruitment, selection and ovulation is essentially granulosa cell proliferation and differentiation, whereas granulosa cell apoptosis represents the degeneration of atresia of the follicle.

Mammalian transcription factor p65 (REL-A) belongs to one of the NF-. Kappa.B (nuclear factor. Kappa.B) family members. The NF-. Kappa.B family consists of five members, NF-. Kappa.B 1 (p 50/p 105), NF-. Kappa.B 2 (p 52/p 100), REL-A, REL-B and REL (cREL). These proteins dimerize to form functional NF κ B. The NF kappa B which is modified and activated after translation is transferred into cell nucleus from cytoplasm and is combined with downstream target genes to regulate the transcription expression of the target genes, and participates in inflammatory reaction, immune response reaction, cell proliferation differentiation and apoptosis and the generation and development of tumors.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides the application of the p65 gene in the porcine ovary granular cells. The expression level of the p65 gene in the granular cells is changed by a genetic engineering technology, and the application of the p65 gene in the pig ovarian granular cell proliferation, apoptosis, estrogen secretion and estrogen signal pathway is determined.

Another purpose of the invention is to provide application of the p65 gene in immature/mature ovaries and follicular granulosa cells with different sizes of sows.

It is still another object of the present invention to provide a small interfering RNA fragment (siRNA) that inhibits the expression of the p65 gene.

The purpose of the invention is realized by the following technical scheme: application of p65 gene in porcine ovarian granulosa cells.

Under the in vitro environment, the p65 gene promotes the proliferation of the ovarian granulosa cells and inhibits the apoptosis of the ovarian granulosa cells.

In an in vitro environment, the p65 gene promotes the ovarian granulosa cells to secrete estrogen.

In an in vitro environment, the p65 gene activates the expression of estrogen signal pathway related genes.

The estrogen signal channel related genes are ESR1, ESR2, HSD3B1, HSD17B4 and CYP19A genes.

The application of the p65 gene in preparing the medicine for promoting the ovarian granulosa cells to secrete estrogen is a mode of increasing the exogenous p65 gene.

The exogenous p65 gene is preferably added by the following method: connecting the p65 gene to a vector to construct a overexpression vector containing the p65 gene; the overexpression vector containing the p65 gene was then transfected into ovarian granulosa cells.

The overexpression vector containing the p65 gene is preferably constructed by the following method:

(1) Extracting RNA of the pig ovarian granulosa cells, performing reverse transcription on the RNA to form cDNA, and performing PCR amplification by using the cDNA as a template to obtain a target fragment;

(2) And connecting the target fragment to a pcDNA3.1 vector which is cut by restriction enzymes Kpn I and Xba I to obtain a recombinant vector, namely the overexpression vector containing the p65 gene.

The primers used for PCR amplification in step (1) are as follows:

Forwad:5’-GGGGTACCATGGACGACCTCTTCCCCCT-3’；

Reverse:5’-GCTCTAGATTAGGAGCTGATCTGACTCA-3’。

application of the p65 gene in immature/mature ovaries of sows and follicular granulosa cells with different sizes.

In an in vitro environment, in immature and mature ovarian tissues, the relative expression amount of mRNA of the p65 gene in the mature ovarian tissue is obviously higher than that of an immature ovarian.

In an in vitro environment, in follicular granulosa cells with different sizes, the relative expression quantity of mRNA of a p65 gene in the granulosa cells of large follicles (more than or equal to 5 mm) is obviously higher than that of small follicles (less than or equal to 3 mm).

A small interfering RNA (siRNA) for inhibiting p65 gene expression is si-p65, and the targeting nucleotide sequence is as follows:

si-p65 targeting nucleotide sequence: 5 'GCACCGGATTGAGGAGAAA-3'.

The application of the small interfering RNA segment (siRNA) for inhibiting the expression of the p65 gene in the porcine ovarian granular cells is in-vitro environment.

The verification results of the invention are as follows:

1. the invention detects that the relative expression quantity of mRNA of the P65 gene in the mature ovarian tissue of the pig is obviously higher (P < 0.05) than that of the immature ovarian by a qRT-PCR method (figure 1). These results suggest that the expression of the p65 gene may be involved in the maturation of the ovaries of sows.

2. The invention detects that the relative expression quantity of mRNA of the P65 gene in the porcine large follicle (more than or equal to 5 mm) granulosa cells is obviously higher (P is less than 0.01) than that of small follicles (less than or equal to 3 mm) by a qRT-PCR method (figure 2). These results suggest that the expression of the p65 gene may be involved in the development of ovarian follicles.

3. According to the invention, after the P65 gene is over-expressed in the granular cell, the proliferation rate of the granular cell is obviously increased (P < 0.05) compared with that of a control group (pcDNA3.1) through an EdU method (FIG. 3 a), and after the expression of the P65 gene is interfered, the proliferation rate of the granular cell is obviously reduced (P < 0.01) compared with that of a control group (NC) (FIG. 3 b).

4. The invention detects through Annexin V-FITC/PI technology that after the P65 gene is over-expressed in the granular cell, the apoptosis rate of the granular cell is obviously reduced (P < 0.05) compared with a control group (pcDNA3.1) (figure 4 a), and after the expression of the P65 gene is interfered, the apoptosis rate of the granular cell is obviously increased (P < 0.01) compared with the control group (NC) (figure 4 b).

5. According to the invention, after the P65 gene is over-expressed in the granular cell, the estrogen secretion amount of the granular cell is obviously increased (P < 0.01) compared with that of a control group (pcDNA3.1) through ELISA technology (figure 5 a), and after the expression of the P65 gene is interfered, the estrogen secretion amount of the granular cell is not obviously changed compared with that of the control group (NC) (figure 5 b).

6. According to the invention, after overexpression and p65 gene interference in granular cells are detected by qRT-PCR, the relative mRNA expression amounts of ESR1, ESR2, HSD3B1, HSD17B4 and CYP19A genes related to estrogen signal channel are obviously changed (figure 6).

The invention takes a p65 Gene (Gene ID: 100135665) as a research object, adopts a molecular cell biology method to research the application of the Gene in the porcine ovarian granulosa cells, proves the application of the p65 Gene in the porcine ovarian granulosa cells, proves that the p65 Gene can participate in promoting the proliferation and the estrogen secretion of the porcine ovarian granulosa cells and inhibiting the apoptosis of the porcine ovarian granulosa cells through over-expression and interference of the p65 Gene, and further indicates that the p65 Gene can participate in promoting the development and maturation of ovarian follicles.

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

the invention detects the relative expression quantity of p65 mRNA in immature and mature ovarian tissues of pigs and follicular granulosa cells with different sizes, detects the proliferation, apoptosis and estrogen secretion conditions of the granulosa cells after overexpression and interference of p65, and is a molecular mechanism accumulation material in the ovarian follicular development process of sows. Meanwhile, the results of the invention show that the p65 gene participates in promoting the proliferation and the estrogen secretion of granulosa cells, activates an estrogen signal channel, and regulates the expression of related genes ESR1, ESR2, HSD3B1, HSD17B4 and CYP 19A.

Drawings

FIG. 1 is a statistical graph of the relative expression of p65 mRNA in porcine immature and mature ovarian tissues.

FIG. 2 is a statistical graph showing the relative expression amounts of p65 mRNA in large and small vacuolar granular cells.

FIG. 3 is a graph showing the results of the EdU method for detecting granulosa cell proliferation after overexpression and interference with p 65; wherein, the graph a is the proliferation condition of the granular cells after the overexpression of p65 is detected by an EdU method; FIG. b shows the proliferation of granulosa cells after detecting interference with p65 by the EdU method.

FIG. 4 is a graph showing the results of granular cell apoptosis after detecting overexpression and interference with p65 by Annexin V-FITC; wherein, the picture a is the apoptosis condition of granular cells after the Annexin V-FITC method detects the over-expression of p 65; panel b shows the apoptosis of granulosa cells after interfering with p65 by Annexin V-FITC.

FIG. 5 is a graph showing the results of ELISA to detect estrogen secretion from granulosa cells after overexpression and interference with p 65; wherein, the graph a is the secretion of estrogen in granulosa cells after the overexpression of p65 is detected by an ELISA method; FIG. b shows the ELISA method for detecting the estrogen secretion in granulosa cells after the p65 overexpression.

FIG. 6 is a statistical graph of the relative expression amount of mRNA of estrogen signaling pathway related genes in granulosa cells after overexpression and interference with p 65; wherein, the graph a shows the relative expression quantity of mRNA of the estrogen signal channel related gene in the granulosa cells after over-expression of p 65; FIG. b shows the relative expression level of mRNA of estrogen signaling pathway-related gene in granulosa cells after p65 interference.

Detailed Description

The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto. It should be understood that the embodiments described in this specification are only for the purpose of illustrating the invention and are not to be construed as limiting the invention, and the parameters, proportions and the like of the embodiments may be suitably selected without materially affecting the results. The examples are reagents and process steps conventional in the art, except as otherwise indicated. Unless otherwise specified, reagents and starting materials for use in the invention are commercially available.

In the present invention, the results of 3 independent experiments in each example were analyzed by statistical methods, and the "mean ± standard deviation" was calculated, and the analysis of significance of difference was performed by one-way analysis of variance (in the figure, "+" indicates P <0.05, and "+" indicates P < 0.01).

Example 1 construction of p65 Gene overexpression vectors

Analysis by BioEdit software revealed that the CDS region sequence of the p65 gene had no restriction enzyme sites for Kpn I and Xba I, while the vector pcDNA3.1 (available from Invitrogen, inc., cat. No. V79020) had Kpn I and Xba I. primer premier 5.0 software designs a p65 gene CDS region primer, and Kpn I and Xba I enzyme cutting site sequences (Forwad: 5-. Extracting RNA of the pig ovarian granular cells, performing reverse transcription to form cDNA, performing PCR amplification on a target fragment by using the pig ovarian granular cell cDNA as a template, performing purification recovery, performing double enzyme digestion, connecting a pcDNA3.1 vector, performing transformation, screening, sequencing and identifying to obtain endotoxin-free plasmids (the endotoxin-free plasmid small quantity extraction kit is purchased from magenta company), and naming the endotoxin-free plasmids as pcDNA3.1-p65.

EXAMPLE 2 culture of ovarian granulosa cells

(1) Putting ovary tissues collected in a slaughterhouse into PBS (phosphate buffer solution) or physiological saline containing 1% (w/w) double antibody, putting on ice, and rapidly bringing back to a laboratory;

(2) Cleaning the collected ovary for 3 times by PBS or normal saline (containing 1% (w/w) double antibody) in an aseptic culture room, quickly transferring to a super-clean workbench, and shallowly inserting into the ovarian follicle with a cavity by using a 1mL aseptic disposable syringe to absorb follicular fluid;

(3) Placing the follicular fluid into a centrifuge tube containing a proper amount of DMEM, and centrifuging at 800rpm at room temperature for 5min;

(4) Discarding the supernatant, resuspending with DMEM, centrifuging, and repeatedly washing the cells for 2 times; preparing a DMEM complete culture medium: 89% (w/w) high-glucose DMEM +10% (w/w) FBS +1% (w/w) double antibody;

(5) Suspending the cells with complete medium, and inoculating the cells in a 75mL culture flask; standing at 37 deg.C, 5% CO ₂ And (5) standing and culturing in an incubator.

The double-resistance is penicillin and streptomycin.

Example 3 inoculation and transfection of ovarian granulosa cells

(1) When the confluence degree of the granular cells reaches about 70-90%, abandoning the culture medium, and washing the cells for 3 times by using preheated PBS;

(2) Adding 0.25% trypsin for digestion, placing in an incubator for about 3min, observing under a microscope until most cells float, and immediately adding equivalent stop solution (complete culture medium) to stop digestion;

(3) Washing with PBS for 2 times, and centrifuging at 800rpm for 5min;

(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 70-90%;

(6) Transfection method according to Invitrogen

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

(7) The transfected cells were placed at 37 ℃ and 5% CO ₂ Continuously culturing in an incubator;

(8) Cells were harvested 24 or 48h after transfection depending on the purpose of the experiment.

Example 4RNA extraction reverse transcription

The extraction of total RNA of cells refers to the operating instruction of TRIzol of Takara company, and the specific operating steps are as follows:

(1) Grinding the ovarian tissues by using liquid nitrogen, adding 1mL of TRIzol into each 50-100 mg of the ovarian tissues, and repeatedly blowing and beating for several times; extracting total RNA from adherent granulosa cells at a rate of 10cm per cell ² 1mL of TRIzol is directly added to the bottom area of the cell culture plate;

(2) Standing on ice for 10min to fully lyse the tissue/cells, centrifuging at 12000rpm for 5min, discarding the precipitate, and taking the supernatant into a new 1.5mL RNase-free tube;

(3) Adding 200 mu L chloroform (1 mL TRIzol) and violently shaking for 15-30 s, standing on ice for 15min, and centrifuging at 12000rpm at 4 ℃ for 15min;

(4) Absorbing the upper aqueous phase and placing the upper aqueous phase in a new 1.5mL RNase-free EP tube;

(5) Adding 500uL isopropanol (1 mL of TRIzol), slightly turning upside down, mixing, standing on ice for 10min, and centrifuging at 4 deg.C and 12000rpm for 10min;

(6) Discarding the supernatant, placing at room temperature, adding 1mL of 75% ethanol-DEPC along the tube wall to wash RNA, centrifuging at 12000rpm at 4 ℃ for 5min, and discarding the supernatant;

(7) Vacuum drying for 5-10 min, and avoiding excessive drying of RNA precipitate;

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

mRNA reverse transcription was performed with reference to PrimeScriptTM RT Master Mix (Perfect Real Time) cDNA reverse transcription kit from TaKaRa.

Example 5qRT-PCR

In the invention, the relative expression quantity of the target gene is detected by a Maxima SYBR Green qPCR Master Mix (2X) kit (Thermo Scientific company). Experimental results 2 ^-ΔΔCt The relative expression level of the target gene was calculated by the method (the calculation formula is shown below).

Relative expression level of target gene =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 gene, and qRT-PCR primers used by the invention are as follows:

qRT-PCR-p65Forward：5′-CATGCGCTTCCGCTACAAG-3′；

Reverse：5′-GGTCCCGCTTCTTTACACAC-3′；

qRT-PCR-ESR1Forward：5′-TTACCTGGAGAATGAGCCGAG-3′；

Reverse：5′-GGCCATGCTTCCCTTGTCA-3′；

qRT-PCR-ESR2Forward：5′-GATGCCTTGGTCTGGGTGAT-3′；

Reverse：5′-AGTGTTCCGTGCCCTTGTTA-3′；

qRT-PCR-HSD3B1Forward：5′-GGGGCTTCTGCCTTGATTCCA-3′；

Reverse：5′-GGTTTTCAGCGCCTCCTTGTGC-3′；

qRT-PCR-HSD17B4Forward：5′-CCCAACGCAGGAGACTTAAAAT-3′；

Reverse：5′-CCAGAGCCCATAACGAAGACAGA-3′；

qRT-PCR-CYP19AForward：5′-GCTGGACACCTCTAACAACCTCTT-3′；

Reverse：5′-TTGCCATGCATCAAAATAACCCT-3′；

qRT-PCR-GAPDH Forward：5′-TCGGAGTGAACGGATTTG-3′；

Reverse：5′-TCACCCCATTTGATGTTGG-3′。

example 6 granulosa cell proliferation assay

The experiment for detecting Cell proliferation by the EdU method in the invention refers to Cell-Light of Ruibo Biotech Co., ltd, guangzhou City ^TM EdU Apollo 567In vitro Kit. The specific steps are as follows (taking a 96-well plate as an example):

(1) Diluting an Edu solution by using a cell culture medium according to the volume ratio of 1000 to 1 to prepare a proper amount of 50 mu M Edu culture medium;

(2) Adding 100 mu L of 50 mu M Edu culture medium into each hole, incubating for 2 hours, and removing the culture medium;

(3) Washing the cells with PBS for 1-2 times, 5 minutes each time;

(4) Adding 50 μ L of cell fixing solution (PBS containing 4% paraformaldehyde) into each well, incubating for 30min at room temperature, and discarding the fixing solution;

(5) Adding 50 mu L of 2mg/mL glycine into each hole, and after incubating for 5 minutes by a decoloring shaker, removing the glycine solution;

(6) Adding 100 mu L PBS into each hole, washing for 5 minutes by a decoloring shaker, and discarding the PBS;

(7) Adding 100 mu L of 1 XApollo staining reaction solution into each hole, incubating for 30 minutes in a light-proof, room temperature and decolorizing shaker, and then discarding the staining reaction solution;

(8) Adding 100 μ L of penetrant (PBS (0.5%; triton X)) and washing with decolorizing shaker for 2-3 times (5 min each time), and discarding the penetrant;

(9) Diluting a reagent F by using a deionized water bank 100;

(10) Adding 100 mu L of 1 × Hoechst3342 reaction liquid into each hole, incubating for 30 minutes in a dark place at room temperature and a decoloring shaking table, and then removing the dyeing reaction liquid;

(11) Adding 150 mu L PBS into each hole to wash for 1-3 times;

(12) Adding 100 mu L PBS into each hole for storage;

(13) After the staining was completed, a photograph was taken with a fluorescence microscope.

Example 7 granular cell apoptosis assay

The invention uses Annexin V-FITC/PI technology to detect Apoptosis, and the specific operation steps are carried out according to the specification of FITC Annexin V Apoptosis Detection Kit with PI Kit of Guangzhou Korea Biotechnology Limited company:

(1) Placing the cell culture plate at room temperature, gently rinsing cells in the culture plate by using PBS, and removing the PBS;

(2) Adding 0.25% trypsin for digestion, placing in an incubator for about 3min, observing most cells floating under a microscope, and immediately adding an equal amount of stop solution (complete culture medium) to stop digestion.

(3) Cells were harvested by centrifugation at 1000rpm for 5min, the supernatant discarded, and the cells were washed twice with pre-chilled PBS. Adjusting the number of cells per tube to 0.2-1.0 x 10 ⁶ 400 μ L of 1-fold Buffer was added to resuspend the cells.

(4) mu.L of FITC-Annexin V was added to each tube in turn, and the mixture was reacted for 15min at room temperature (25 ℃) in the dark.

(5) Then sequentially adding 10 mu L of PI, lightly mixing uniformly, and reacting for 5min at 4 ℃ in a dark place.

(6) Immediately after the reaction, the reaction solution was analyzed by flow cytometry.

Example 8 detection of Estrogen in granular cell culture supernatant

The invention uses ELISA method to detect the estrogen secretion level of granulocytes, the concrete operation steps refer to the specification of pig estrogen (E) enzyme-linked immunosorbent assay (ELISA) kit of Jiangsu Bo-Shen biological technology limited company:

(1) Collecting cell culture solution supernatant by using an aseptic centrifuge tube, centrifuging at 3000rpm for 20min, and removing precipitate;

(2) A standard well and a sample well are provided. Adding standard substance with different concentrations into standard substance wells respectively to obtain 50 μ L standard substance; the sample holes are respectively provided with a blank hole and a sample hole to be detected, 40 mu L of sample diluent is added into the sample hole to be detected of the enzyme-labeled coated plate, and 10 mu L of sample to be detected is added (the final dilution of the sample to be detected is 5 times);

(3) Incubating at 37 ℃ for 30min, and then removing liquid in the ELISA plate;

(4) Filling each hole with a washing solution, standing for 30s, discarding, and washing for 5 times;

(5) Adding 50 mu L of enzyme-labeled reagent into each hole except for blank holes;

(6) Incubation and washing are carried out in the same steps 3 and 4;

(7) Adding 50 μ L of color-developing agent A and B into each well, mixing, and developing at 37 deg.C in dark for 15min;

(8) Stop solution 50. Mu.L per well to stop the reaction (at this time, the solution in the well changes from blue to yellow);

(9) The stop solution was added within 15min, and the absorbance was measured at a wavelength of 450nm with the blank well being zeroed.

Analysis of results

1. Before the ovaries of the sows are not completely mature, a plurality of small follicles (the diameter is less than or equal to 3 mm) protruding out of the surfaces are formed, and the ovaries are similar to mulberries; at the first estrus, the ovary matures, many follicles of different sizes (follicles with a diameter of 5mm or more) protrude from the surface of the ovary, and there is a sign of ovulation. Therefore, the cDNA of the immature ovarian tissues (about 160-day old large two-way sow) and the mature ovarian tissues (about 200-day old large two-way sow) of the pig are taken as templates, and the relative expression level of the mRNA of the P65 gene in the mature ovarian tissues of the pig is detected to be remarkably higher than that of (P < 0.05) immature ovarian tissues by a qRT-PCR method (primers are shown in example 5 as qRT-PCR-P65) (figure 1). These results suggest that the p65 gene may be involved in promoting the maturation of the ovaries of sows.

The immature and mature ovarian tissues of the pigs are from big and large binary mixed sows in Yangjiang Baojun pig farms.

2. After the ovaries of the sows are mature, the ovulation process of the sows is changed periodically, and follicles with different sizes appear on the surfaces of the ovaries. Therefore, the ovarian follicles are classified into two grades of large follicles (more than or equal to 5 mm) and small follicles (less than or equal to 3 mm) according to the diameter, and the mRNA expression change of the pig P65 gene is detected by a qRT-PCR method (primers are shown in example 5 qRT-PCR-P65) by taking cDNA of the pig large follicles (more than or equal to 5 mm) and the small follicles (less than or equal to 3 mm) as a template, so that the relative expression quantity of the mRNA of the P65 gene in the pig large follicles (more than or equal to 5 mm) is remarkably higher than that of (P < 0.01) the small follicles (less than or equal to 3 mm) (figure 2). These results suggest that the p65 gene may be involved in promoting follicular development.

The ovarian follicles are from healthy commercial sows (Du long three-way hybrid pigs) in a Hope holly slaughter house in Guangzhou city.

3. After healthy commercial sow ovaries are collected from slaughterhouses, ovarian granulosa cells are isolated and cultured. The invention exogenously synthesizes a p65 gene over-expression vector (pcDNA3.1-p 65) and a small interfering RNA fragment (si-p 65) by a genetic engineering technology, transfects the p65 gene over-expression vector and the small interfering RNA fragment into a pig ovary particle cell (taking a transfection vector pcDNA3.1 and NC as a contrast), and detects the proliferation condition of the particle cell after 24 hours by referring to an EdU kit instruction. The detection results show that after the P65 gene is over-expressed, the proliferation rate of the granular cells is remarkably increased (P < 0.05) compared with the control group (pcDNA3.1) (FIG. 3 a), and after the P65 gene expression is interfered, the proliferation rate of the granular cells is remarkably reduced (P < 0.01) compared with the control group (NC) (FIG. 3 b). Wherein the content of the first and second substances,

small interfering RNA fragment (si-p 65) targeting sequence: 5 'GCACCGGATTGAGAGAAA-3'.

The small interference RNA fragment is synthesized by Ruibo biotechnology, inc., guangzhou city; control NC was from the company lunbo biotechnology limited, guangzhou (NC is a universal negative control product of the company, the same applies below).

4. After healthy commercial sow ovaries are collected from a slaughterhouse, ovarian granulosa cells are isolated and cultured. The invention exogenously synthesizes a p65 gene overexpression vector (pcDNA3.1-p 65) and a small interference fragment (si-p 65) by a genetic engineering technology, transfects the p65 gene overexpression vector and the small interference fragment into a swine ovarian granular cell, and detects the Apoptosis condition of the granular cell by referring to FITC Annexin V Apoptosis Detection Kit with PI specification after 48 hours (the transfection vectors pcDNA3.1 and NC are used as a reference). The detection result shows that the apoptosis rate of the granular cells is remarkably reduced (P < 0.05) compared with that of a control group (pcDNA3.1) after the P65 gene is over-expressed (figure 4 a), and the apoptosis rate of the granular cells is remarkably increased (P < 0.01) compared with that of the control group (NC) after the P65 gene expression is interfered (figure 4 b).

5. After healthy commercial sow ovaries are collected from a slaughterhouse, ovarian granulosa cells are isolated and cultured. The invention exogenously synthesizes a p65 gene over-expression vector (pcDNA3.1-p 65) and a small interference fragment (si-p 65) by a genetic engineering technology, transfects the p65 gene over-expression vector and the small interference fragment into a swine ovary particle cell, and detects the estrogen secretion condition of the particle cell by referring to an ELISA kit instruction after 48 hours (the transfection vectors pcDNA3.1 and NC are used as a contrast). The detection result shows that after the P65 gene is over-expressed, the estrogen secretion amount of the granular cell is obviously increased (P < 0.01) compared with that of a control group (pcDNA3.1) (figure 5 a), and after the P65 gene expression is interfered, the estrogen secretion amount of the granular cell is not obviously changed compared with that of the control group (NC) (figure 5 b).

6. After healthy commercial sow ovaries are collected from slaughterhouses, ovarian granulosa cells are isolated and cultured. The invention exogenously synthesizes a p65 gene overexpression vector (pcDNA3.1-p 65) and a small interference fragment (si-p 65) by a genetic engineering technology, transfects the p65 gene overexpression vector and the small interference fragment into a pig ovary particle cell, detects the expression quantity of the mRNA of the estrogen signal channel related gene in the particle cell by a qRT-PCR method after 48 hours (the qRT-PCR primer is shown in an example 5), and takes the transfection vectors pcDNA3.1 and NC as a contrast. The detection result shows that after the p65 gene is over-expressed and interfered, the relative mRNA expression quantity of genes ESR1 (primer: qRT-PCR-ESR 1), ESR2 (primer: qRT-PCR-ESR 2), HSD3B1 (primer: qRT-PCR-HSD3B 1), HSD17B4 (primer: qRT-PCR-HSD17B 4) and CYP19A (primer: qRT-PCR-CYP 19A) related to the estrogen signal channel is obviously changed (figure 6).

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> south China university of agriculture

Application of <120> p65 gene in porcine ovarian granulosa cells

<160> 17

<170> SIPOSequenceListing 1.0

<210> 1

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Forwad

<400> 1

ggggtaccat ggacgacctc ttccccct 28

<210> 2

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Reverse

<400> 2

gctctagatt aggagctgat ctgactca 28

<210> 3

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> si-p65 targeting nucleotide sequences

<400> 3

gcaccggatt gaggagaaa 19

<210> 4

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> qRT-PCR-p65 Forward

<400> 4

catgcgcttc cgctacaag 19

<210> 5

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> qRT-PCR-p65 Reverse

<400> 5

ggtcccgctt ctttacacac 20

<210> 6

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> qRT-PCR-ESR1 Forward

<400> 6

ttacctggag aatgagccga g 21

<210> 7

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> qRT-PCR-ESR1 Reverse

<400> 7

ggccatgctt cccttgtca 19

<210> 8

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> qRT-PCR-ESR2 Forward

<400> 8

gatgccttgg tctgggtgat 20

<210> 9

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> qRT-PCR-ESR2 Reverse

<400> 9

agtgttccgt gcccttgtta 20

<210> 10

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> qRT-PCR-HSD3B1 Forward

<400> 10

ggggcttctg ccttgattcc a 21

<210> 11

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> qRT-PCR-HSD3B1 Reverse

<400> 11

ggttttcagc gcctccttgt gc 22

<210> 12

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> qRT-PCR-HSD17B4 Forward

<400> 12

cccaacgcag gagacttaaa at 22

<210> 13

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> qRT-PCR-HSD17B4 Reverse

<400> 13

ccagagccca taacgaagac aga 23

<210> 14

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> qRT-PCR-CYP19A Forward

<400> 14

gctggacacc tctaacaacc tctt 24

<210> 15

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> qRT-PCR-CYP19A Reverse

<400> 15

ttgccatgca tcaaaataac cct 23

<210> 16

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> qRT-PCR-GAPDH Forward

<400> 16

tcggagtgaa cggatttg 18

<210> 17

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> qRT-PCR-GAPDH Reverse

<400> 17

tcaccccatt tgatgttgg 19

Claims (3)

1. The application of the p65 gene in the swine ovary granular cells is characterized in that: in an in vitro environment, the exogenous p65 gene is added to promote the pig ovarian granulosa cells to secrete estrogen.
2. The application of the p65 gene in the preparation of the medicine for promoting the pig ovarian granulosa cells to secrete estrogen is characterized in that: the exogenous p65 gene is added to promote the pig ovarian granulosa cells to secrete estrogen.
3. 3. The use of claim 1 or 2, wherein the addition of the exogenous p65 gene is achieved by:

   connecting the p65 gene to a vector to construct a overexpression vector containing the p65 gene; the overexpression vector containing the p65 gene was then transfected into porcine ovarian granulosa cells.

Images