Application of ZFP42 gene in porcine ovarian granulosa cells

Abstract

The present invention discloses the application of ZFP42 gene in pig ovarian granulosa cells, and belongs to the technical field of cell engineering and genetic engineering. The present invention takes ZFP42 gene as the starting point, and adopts cell biology method to study its effect on autophagy and apoptosis of pig ovarian granulosa cells. After overexpression of ZFP42, the level of cell autophagy is significantly increased, and after knocking down ZFP42, the level of autophagy is significantly reduced; after overexpression of ZFP42, the apoptosis rate of cells is significantly increased, and after knocking down ZFP42, the apoptosis rate is significantly decreased. The technical scheme of the present invention is well designed and the results are reliable. It is confirmed that ZFP42 promotes autophagy and apoptosis of pig ovarian granulosa cells, which has great application value for studying the mechanism of influence on ovarian follicle development and sow reproductive performance. It provides technical support for improving the fertility of sows, and at the same time has important economic value for the actual production of pig farming.

Description

Technical Field

[0001] The invention belongs to the technical field of cell engineering and gene engineering, and specifically relates to the application of ZFP42 gene in pig ovarian granulosa cells. Background Art

[0002] The reproductive performance of sows is crucial to the production efficiency and economic benefits of modern pig farms. Selecting sows with a high ovulation number can increase the litter size of sows, and the litter size is one of the main objective traits to improve the efficiency of pig breeding. Ovarian GCs regulate the maturation of mammalian oocytes and are an important model for studying the development of follicles. There are two types of ovarian GCs, the outer parietal GCs and the inner cumulus cells. The inner cumulus cells directly surround the oocyte and support the development of the oocyte. Parietal GCs have important endocrine functions and become luteal cells after ovulation, which are considered to be meiotic inhibition signals.

[0003] ZFP42 is located on the negative strand of chromosome 17 of pigs, spanning approximately 7Kb. It has three transcripts, each with approximately 2 to 4 exons. The ZFP42 gene encodes a protein containing four zinc finger structures (C2H2). C2H2 zinc fingers are two conserved cysteines (Cys) and histidine (His) that form a tetrahedral structure that fixes zinc ions. C2H2 zinc fingers consist of two short β strands, followed by an α helix. The amino-terminal part of the helix binds to the main groove in DNA that binds zinc fingers. ZFP42 is produced by the retroposition of YY1 and has a C2H2 protein structure that is highly similar to Yy1. Kristensen et al. detected the expression of ZFP42 in human gonadal development specimens by IHC and found that ZFP42 was mainly expressed in meiotic cells during oogenesis. In addition, ZFP42 targets multiple genes linked to the X chromosome, and there is an evolutionary connection between the reactivation of the X chromosome and pluripotency. Endogenous retroviral elements (ERVs) account for about 10% of the mammalian genome. In contrast to exogenous retroviruses, ERVs are an integral part of the genome in all cells of an organism. Retroviral elements frequently appear in transcripts of mature oocytes. However, the association between the ZFP42 gene and the growth and development of sow ovarian granulosa cells has not been reported. Summary of the invention

[0004] The purpose of the present invention is to provide application of ZFP42 gene in pig ovarian granulosa cells.

[0005] In this study, porcine ZPF42 overexpression vectors were constructed and interference fragments were synthesized. The expression efficiency of ZFP42 gene after treating porcine ovarian granulosa cells with overexpression vectors and interference fragments was detected by qPCR. After treating cells with appropriate concentrations, mRFP-GFP-LC3 adenovirus was used to explore the effect of ZFP42 on cellular autophagy flow; qPCR and Western blot experimental methods were used to explore the expression of mRNA and protein of genes in the cell autophagy-related pathway by ZFP42; Annexin V / PI experimental methods were used to explore the effect of ZFP42 on apoptosis of porcine ovarian granulosa cells. qPCR and Western blot experimental methods were used to explore the expression of mRNA and protein of genes in the cell apoptosis-related pathway by ZFP42.

[0006] The purpose of the present invention is achieved through the following technical solutions:

[0007] The application of increasing exogenous ZFP42 gene to promote autophagy and / or apoptosis of pig ovarian granulosa cells in vitro is achieved by gene overexpression technology.

[0008] The invention discloses an application of inhibiting ZFP42 gene expression to inhibit autophagy and / or apoptosis of pig ovarian granulosa cells in vitro, wherein the inhibition of ZFP42 gene expression is achieved by RNA interference technology.

[0009] Furthermore, the gene overexpression vector used in the gene overexpression technology is prepared by the following method:

[0010] (1) extracting DNA from pig ovarian granulosa cells, and performing PCR amplification using the DNA as a template to obtain the target fragment;

[0011] (2) The target fragment was connected to the pcDNA3.1(+) vector digested with restriction endonucleases XbaI and kpnl to obtain a recombinant vector.

[0012] Furthermore, the primers used for PCR amplification in step (1) are as follows:

[0013] ZFP42 Forward: 5′-GCGCGTTAGCATGGGTGCTTTC-3′;

[0014] ZFP42 Reverse: 5′-CCAGGCATATCTGTCCCCAATCAA-3′.

[0015] Furthermore, the sequence of the small interfering fragment used in the RNA interference technology is as follows:

[0016] si-ZFP42: 5′-CCAAGTGTCGGAGAGAATT-3′.

[0017] Furthermore, the overexpression vector is used at a concentration of 500-1000 ng / mL, and the small interfering fragment is used at a concentration of 50-100 nmol / L.

[0018] Furthermore, the overexpression vector is used at a concentration of 500 ng / mL, and the small interfering fragment is used at a concentration of 50 nmol / L.

[0019] Furthermore, the ZFP42 gene regulates autophagy and / or apoptosis of pig ovarian granulosa cells by interacting with Beclin1 protein.

[0020] Application of an agent for inhibiting ZFP42 gene expression in the preparation of a drug for inhibiting autophagy of pig ovarian granulosa cells, a drug for inhibiting apoptosis of pig ovarian granulosa cells and / or a drug for promoting the development of pig follicles.

[0021] Furthermore, the reagent for inhibiting ZFP42 gene expression is a small interfering fragment, the sequence of which is as follows:

[0022] si-ZFP42: 5′-CCAAGTGTCGGAGAGAATT-3′.

[0023] The verification process of the present invention is as follows:

[0024] (1) A porcine pcDNA3.1(+)-ZFP42 overexpression vector was constructed and named OE-ZFP42; its control group was pcDNA3.1(+) and named OE-NC. An interference fragment of ZFP42 was synthesized and named si-ZFP42; its control group was NC and named si-NC. Finally, the ZFP42 overexpression vector and interference fragment were transfected into porcine ovarian granulosa cells, respectively. After 24 hours, the cells were collected and RNA was extracted for efficiency detection.

[0025] (2) After treating porcine ovarian granulosa cells with mRFP-GFP-LC3 adenovirus, they were transfected with ZFP42 overexpression vectors and interference fragments, respectively, and photographed using a Leica fluorescence microscope 24 hours later. The autophagosomes and autolysosomes of the cells were manually counted, and finally the autophagic flux was evaluated based on the ratio of GFP (green fluorescence) to mRFP (red fluorescence) bright spots.

[0026] (3) Similarly, the ZFP42 overexpression vector and interference fragment were transfected into porcine ovarian granulosa cells, and the cells were collected 24 hours later to extract RNA and protein. The expression of autophagy-related gene mRNA and protein was detected.

[0027] (4) The ZFP42 overexpression vector and interference fragment were transfected into porcine ovarian granulosa cells, respectively. The cells were collected after 24 hours, and the cell apoptosis rate was detected by flow cytometry using the Annexin V / PI method.

[0028] (5) The ZFP42 overexpression vector and interference fragment were transfected into porcine ovarian granulosa cells, and the cells were collected 24 hours later to extract RNA and protein. The expression of apoptosis-related gene mRNA and protein was detected.

[0029] (6) Use the CoIP method to explore the molecular mechanism of ZFP42 regulating autophagy.

[0030] Compared with the prior art, the present invention has the following advantages and effects:

[0031] The technical solution of the present invention is well designed and the results are reliable. In order to confirm the effect of ZFP42 on porcine ovarian granulosa cells, the present invention verifies from multiple levels and angles, and verifies at the cellular level and protein level. The results show that the level of cell autophagy increases significantly after overexpression of ZFP42, and the level of autophagy decreases significantly after knocking down ZFP42; the apoptosis rate of cells increases significantly after overexpression of ZFP42, and the apoptosis rate decreases significantly after knocking down ZFP42.

[0032] The present invention shows for the first time that ZFP42 promotes the expression of mRNA and protein of genes related to autophagy and apoptosis in pig ovarian granulosa cells, thereby affecting cell autophagy and apoptosis. These have great application value in studying the mechanism of influence on ovarian follicle development and sow reproductive performance. It provides technical support for improving sow reproductive capacity and has important economic value for the actual production of pig farming. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 It is a graph showing the results of the efficiency detection of the overexpression vector and the interference fragment; wherein, a is the efficiency detection of the pcDNA3.1(+)-ZFP42 overexpression vector of pig origin; and b is the efficiency detection of the interference fragment of ZFP42.

[0034] Figure 2 These are the results of a study on the effect of ZFP42 on autophagy in pig ovarian granulosa cells; wherein, a is the effect of the overexpression vector and interference fragment of ZFP42 on the cellular autophagic flow detected by autophagy double-labeled adenovirus; b is the effect of the overexpression vector and interference fragment of ZFP42 on the mRNA of autophagy pathway genes detected by RT-qPCR after transfection of the overexpression vector and interference fragment of ZFP42 into the cells, respectively; and c is the effect of the overexpression vector and interference fragment of ZFP42 on the expression of autophagy pathway proteins detected by Western blot after transfection of the overexpression vector and interference fragment of ZFP42 into the cells, respectively.

[0035] Figure 3These are the results of a study on the effect of ZFP42 on apoptosis of pig ovarian granulosa cells; wherein, a is the Annexin V-FITC method used to analyze the apoptosis of pig ovarian granulosa cells; b is the RT-qPCR detection of the effect of transfecting the ZFP42 overexpression vector and interference fragment into the cells, respectively, on the mRNA of apoptosis pathway genes; and c is the Western blot detection of the effect of transfecting the ZFP42 overexpression vector and interference fragment into the cells, respectively, on the expression of apoptosis pathway proteins.

[0036] Figure 4 This is a diagram showing the results of a study on the interaction between ZFP42 and Beclin1. DETAILED DESCRIPTION

[0037] The present invention is further described in detail below in conjunction with embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0038] The experimental methods in the following examples without specifying specific conditions are usually based on conventional conditions.

[0039] In the following examples, statistical methods were applied to analyze the results of 3 independent experiments in each example, and the "mean ± standard deviation" was calculated respectively. The difference significance was analyzed by one-way analysis of variance (in the figure, "*" indicates P < 0.05, and "**" indicates P < 0.01).

[0040] Example 1: Culture of porcine ovarian granulosa cells

[0041] (1) The porcine ovarian granulosa cells of the present invention were collected from a slaughterhouse in Guangzhou. The ovaries of slaughtered sows were collected and stored in a PBS reagent containing 1% double antibiotics (penicillin and streptomycin), and then quickly brought back to the laboratory for processing at low temperature;

[0042] (2) Wash the ovaries several times with PBS containing 1% double antibody and transfer the samples to the cell room;

[0043] (3) Expose the clean bench to ultraviolet light for 20 minutes in advance and wipe it with 75% alcohol;

[0044] (4) Grasp the ovary with forceps and use a 1 mL syringe to draw follicular fluid into 5 mL of DMEM medium. Draw up 9 mL of follicular fluid in each tube.

[0045] (5) Centrifuge at 800 rpm for 5 min, discard the supernatant, add preheated PBS, gently pipette to precipitate cells, and wash twice.

[0046] (6) Cultivate in a 75 mL cell culture flask. First, add 10 mL of complete culture medium (containing 10% calf serum and 1% double antibody) to the culture flask, then add 5 mL of cell suspension and shake gently to mix evenly.

[0047] (7) Place in a 37°C, 5% CO2 incubator and observe the growth of porcine ovarian granulosa cells after 48 hours;

[0048] (8) Wash twice with pre-warmed PBS containing 1% double antibody, and then culture for 24 hours before proceeding with subsequent experiments.

[0049] Example 2: Construction of ZFP42 overexpression vector and interference fragment and efficiency detection

[0050] A. Build:

[0051] (1) DNA was extracted from pig ovarian granulosa cells, and PCR amplification was performed using the DNA as a template to obtain the target fragment of ZFP42 (Gene ID: 100627512);

[0052] (2) The target fragment was connected to the pcDNA3.1(+) vector digested with restriction endonucleases XbaI and kpnl to obtain a recombinant vector.

[0053] The primers used for PCR amplification in step (1) are as follows:

[0054] ZFP42 Forward: 5′-GCGCGTTAGCATGGGTGCTTTC-3′;

[0055] ZFP42 Reverse: 5′-CCAGGCATATCTGTCCCCAATCAA-3′.

[0056] (3) Design and synthesize the small interfering fragment sequence of the ZFP42 gene:

[0057] si-ZFP42: 5′-CCAAGTGTCGGAGAGAATT-3′.

[0058] B. Efficiency test:

[0059] (1) Digest the cultured cells with trypsin and place them in a 37°C, 5% CO2 incubator for 3-5 minutes. When most of the cells are observed to be suspended under a microscope, immediately add an equal amount of complete culture medium to terminate the digestion;

[0060] (2) Collect the cell suspension and centrifuge at 800 rpm for 5 min. Wash the cells twice with preheated PBS, resuspend them in complete culture medium, add the cell suspension evenly into a 12-well plate, shake gently, and culture in a 37°C, 5% CO2 incubator for 24 h.

[0061] (3) Observe the cell status. When the cell confluence reached about 80%, the ZFP42 overexpression vector (OE-ZFP42) and overexpression control (OE-NC) at concentrations of 50 ng / mL, 200 ng / mL and 500 ng / mL were transfected into the cells, and the ZFP42 interference fragment (si-ZFP42) and interference control (si-NC) at concentrations of 50 nmol / L and 100 nmol / L were transfected into the cells, respectively. After 24 hours, the cells were collected to extract RNA, and the content of the ZFP42 gene was detected by RT-qPCR method.

[0062] Example 3: Western Blot

[0063] (1) Total protein extraction from cells (using the P1250 total protein extraction kit from Beijing Prilai Gene Technology Co., Ltd.):

[0064] ① Digest and wash the cells, collect the cells by centrifugation at 800 rpm, and 6 Add 0.5 mL of lysis buffer to the cells, vortex and resuspend, incubate at 4 °C for 2 min;

[0065] ② Add 1 mL of extraction reagent to every 0.5 mL of lysate, shake and mix, and let stand at 4°C for 10 min;

[0066] ③ Centrifuge at 10,000rpm and 4℃ for 10min. The solution will separate into upper and lower phases. Aspirate the upper phase and the lower phase as much as possible, and collect the protein floccules in the middle of the two phases. If the phases cannot be separated, mix again with 50μL of distilled water and centrifuge;

[0067] ④ Add 1 mL of ethanol to wash the precipitate, centrifuge at 10,000 rpm at 4°C for 3 min, remove all liquid in the tube, and dry at room temperature.

[0068] (2)SDS-PAGE:

[0069] ① Use a multifunctional microplate reader to measure the wavelength of A 562 , calculate the average absorbance of each concentration of protein standard, draw the standard curve, calculate the regression equation, and quantify the protein (using the BCA protein quantification kit);

[0070] ② Sample preparation: mix 20 μg total protein with 5× loading buffer at a ratio of 5:1 and boil for 5 min;

[0071] ③ Prepare gel and spot samples. Run the concentrated gel at a voltage of 60-100V for about 30 minutes, and run the separation gel at a voltage of 100-200V for about 60 minutes (stop electrophoresis when bromofen blue runs to about 1-2cm from the bottom of the gel; or adjust the gel running voltage and time according to the specific situation);

[0072] ④ Cut the gel strip containing the target protein according to the molecular weight standard of the protein marker, and select the transfer current of 300mA for 20min;

[0073] ⑤After the transfer, rinse the membrane gently with TBST and block it with 5% skim milk powder at room temperature for 1 to 2 hours;

[0074] ⑥ Gently rinse the membrane with TBST for 6 times, 5 min each time, dilute the primary antibody Histone H3 trimethyl K4 (purchased from Abcam) with TBST 1:5000, and incubate at 4°C overnight;

[0075] ⑦ Gently rinse the membrane with TBST 6 times, 5 min each time, dilute the secondary antibody Goat anti-rabbit IgG-HRP (purchased from SANTA CRUZ) at 1:4000, and incubate at room temperature for about 2 hours;

[0076] ⑧ Gently rinse the membrane with TBST 6 times, 5 min each time, and develop the color using an ECL color development kit;

[0077] ⑨ Observe the brightness of the fluorescence in a darkroom to determine the exposure time, develop and fix the film, take pictures (or scan), and use Image Plus software to analyze the protein bands in the film.

[0078] Example 4: Granulocyte autophagy detection

[0079] The present invention uses the autophagy double-labeled adenovirus (HBAD-mRFP-GFP-LC3) method to detect cell autophagy. Referring to the instructions of the HBAD-mRFP-GFP-LC3-Autophagy Detection Kit of Shanghai Hanheng Biotechnology Co., Ltd., the specific operation steps are as follows:

[0080] (1) Cell preparation: Trypsin-digested cells were seeded into 48-well plates. The cell confluence was between 30% and 50% during virus infection.

[0081] (2) 1 / 2 volume infection: During viral infection, 1 / 2 volume of fresh culture medium was added, and the volume was replenished to the normal culture volume 4 h after adding adenovirus infection. OE-NC, OE-ZFP42, si-NC, and si-ZFP42 were transfected into the cells, respectively.

[0082] (3) Observation and analysis of statistical results: 24 h after infection, cells were fixed, photographed using a confocal microscope, and analyzed using ImageJ.

[0083] Example 5: Granulocyte apoptosis detection

[0084] The present invention uses Annexin V-FITC technology to detect cell apoptosis. Referring to the instructions of the Annexin V-FITC Apoptosis Detection Kit of BioVision, the specific operation steps are as follows:

[0085] (1) Cells were inoculated into 6-well plates and cultured until the confluence reached 50-80%, and then washed with PBS solution;

[0086] (2) Digest the cells with EDTA-free trypsin and wash the cells with 2 mL of PBS solution;

[0087] (3) Take 0.5 mL of cell suspension (about 5×10 5 cells), add 500 μL 1× Binding Buffer;

[0088] (4) Add 5 μL Annexin V-FITC and 5 μL Propidium Iodide at room temperature and incubate at room temperature in the dark for 5 min;

[0089] (5) Immediately analyze the cells using flow cytometry (3 replicates per group).

[0090] Example 6: Co-immunoprecipitation

[0091] The cells were washed twice with PBS and digested. Co-immunoprecipitation (CoIP) was performed according to the instructions of ACE's rProtein A / G Magnetic IP / CO-IP Kit. Finally, the resin was heated at 100°C for 10 min with SDS sample buffer to prepare SDS-PAGE analysis, and the target proteins (ZFP42, Beclin1) were detected by Western Bolt.

[0092] Result analysis:

[0093] 1. Select the optimal overexpression vector and interference fragment concentration for treating porcine ovarian granulosa cells

[0094] The ZFP42 overexpression vector (OE-ZFP42) and overexpression control (OE-NC) at concentrations of 50 ng / mL, 200 ng / mL, and 500 ng / mL were transfected into cells, and the cells were collected and RNA was extracted 24 hours later to detect the content of the ZFP42 gene. The optimal concentration of OE-ZFP42 was finally determined to be 500 ng / mL ( Figure 1 a) in the figure.

[0095] The cells were transfected with 50nmol / L and 100nmol / L concentrations of ZFP42 interference fragment (si-ZFP42) and interference control (si-NC), respectively. After 24 hours, the cells were collected to extract RNA and the content of ZFP42 gene was detected. Finally, it was determined that si-ZFP42-3 had a better interference effect, and its most suitable concentration was 50nmol / L ( Figure 1 b) in the above.

[0096] 2. Effect of ZFP42 on autophagy in porcine ovarian granulosa cells

[0097] In order to study the effects of OE-ZFP42 and si-ZFP42 on autophagy in porcine ovarian granulosa cells, the cells were transfected and treated respectively, and the autophagy fluorescence double-labeled adenovirus was used to analyze the generation of cellular autophagic flux.

[0098] Results of fluorescent double-labeled adenovirus Figure 2 Figure a) shows that compared with OE-NC, the number of red fluorescence increased significantly after overexpression of ZFP42 (P<0.05); compared with si-NC, the number of red fluorescence decreased significantly after knockdown of ZFP42 (P<0.05). This result indicates that the ZFP42 gene promotes autophagic flux in GCs.

[0099] 3. Effect of ZFP42 on the mRNA and protein expression of autophagy-related genes in porcine ovarian granulosa cells

[0100] To study the effects of OE-ZFP42 and si-ZFP42 on the mRNA and protein expression of autophagy-related genes in porcine ovarian granulosa cells, cells were transfected and treated respectively. The mRNA of autophagy-related genes was quantified by RT-qPCR. qPCR results ( Figure 2 b) found that after overexpression of ZFP42, the expression levels of ATG3, ATG7, ATG12, ATG16L, Beclin1, mTOR and LC3b genes were significantly increased (P<0.05). After knocking down ZFP42, the expression levels of these autophagy-related gene mRNAs were significantly decreased (P<0.05). The results show that ZFP42 promotes the expression of autophagy-related gene mRNAs in cells.

[0101] The Western Blot method was used to quantify autophagy-related proteins. Figure 2 c) found that after overexpression of ZFP42, the expression of ATG5, ATG7, Beclin1, mTOR and LC3b proteins increased significantly (P<0.05). After knocking down ZFP42, the expression of these autophagy-related proteins decreased significantly (P<0.05). The results show that ZFP42 promotes the expression of autophagy-related proteins in cells.

[0102] 4. Effect of ZFP42 on apoptosis of porcine ovarian granulosa cells

[0103] After the ZFP42 gene induces cell autophagy, will it further induce cell apoptosis? First, Annexin V-FITC was used to detect the cell apoptosis rate. The results were ( Figure 3 In a), it was found that after overexpression of ZFP42, the apoptosis rate of GCs was significantly increased (P<0.05). After knocking down ZFP42, the apoptosis rate of GCs was significantly decreased.

[0104] The mRNA of apoptosis-related genes was quantified using RT-qPCR. Figure 3 b) found that apoptosis-related genes such as Caspase3, Caspase8, and Caspase9 were significantly increased after overexpression of ZFP42 (P<0.05), and significantly decreased after knockdown of ZFP42 (P<0.05). The results show that ZFP42 promotes the expression of apoptosis-related gene mRNA in cells.

[0105] Western Blot method was used to quantify apoptosis-related proteins. Figure 3 In c), it was found that the apoptosis-related proteins Caspase8 and Caspase9 were significantly increased after overexpression of ZFP42, and significantly decreased after knockdown of ZFP42 (P<0.05). The results showed that ZFP42 promoted the expression of apoptosis-related proteins in cells.

[0106] 5. ZFP42 interacts with Beclin1 protein

[0107] Through CoIP experiments, the molecular mechanism of ZFP42 gene regulating cell autophagy was further studied. First, the JASPAR website predicted that the transcription factor ZFP42 might bind to the autophagy gene Beclin1. ZFP42 antibody was CoIPed with pig ovary total protein, and the protein complex after CoIP was collected and denatured. Finally, the expression of Beclin1 (52KD) protein was detected by Western Bolt. Results ( Figure 4 ) found that ZFP42 interacts with Beclin1 protein, and ZFP42 may regulate cellular autophagy by regulating Beclin1.

[0108] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. The use of inhibiting ZFP42 gene expression to inhibit autophagy and / or apoptosis of pig ovarian granulosa cells in vitro, characterized in that: The inhibition of ZFP42 gene expression is achieved through RNA interference technology.

2. The use according to claim 1, characterized in that: The small interfering fragment sequence used in the RNA interference technology is as follows: si-ZFP42:5'-CCAAGTGTCGGAGAGAATT-3'.

3. The use according to claim 2, characterized in that: The small interfering fragment is used at a concentration of 50 to 100 nmol / L.

4. The use according to any one of claims 1 to 3, characterized in that: The ZFP42 gene regulates autophagy and / or apoptosis of pig ovarian granulosa cells by interacting with Beclin1 protein.

5. Use of an agent for inhibiting ZFP42 gene expression in the preparation of a drug for inhibiting autophagy of pig ovarian granulosa cells, a drug for inhibiting apoptosis of pig ovarian granulosa cells and / or a drug for promoting the development of pig follicles, characterized in that: The reagent for inhibiting ZFP42 gene expression is a small interfering fragment, and the sequence is as follows: si-ZFP42:5'-CCAAGTGTCGGAGAGAATT-3'.

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