Application of DLAT gene in regulation of granular cell proliferation and / or apoptosis in follicular development

Abstract

The invention discloses application of a DLAT gene to regulation of granular cell proliferation and / or apoptosis in follicular development. According to the invention, DLAT is taken as a research object, and application of DLAT in ovarian granular cells is researched by adopting molecular and cell biology methods: by increasing the degree of pyruvic acid, it is found that the increase of pyruvic acid can promote transcription of DLAT genes. Through overexpression or interference of DLAT, DLAT can promote proliferation of ovarian granular cells and inhibit apoptosis. The DLAT can promote follicle development by infecting pig follicles with DLAT lentiviruses. DLAT lentivirus injection shows that DLAT can promote development of ovarian follicles of mice and promote oestrus. In addition, the combination of the DLAT protein and the proteins of the CASP3 and the CASP9 is found in granulosa cells and mouse ovary. The method has a good application value for researching the development mechanism of ovarian follicles and the like.

Description

Technical Field

[0001] The present invention belongs to the technical fields of cell engineering and genetic engineering, and particularly relates to the application of DLAT gene in regulating granulosa cell proliferation and / or apoptosis in follicular development. Background Art

[0002] In mammals, follicular growth and development is an extremely complex physiological process, which is closely related to the reproductive performance of livestock, and its reproductive efficiency further affects production performance. Granulosa cells play a key role in the growth and development of oocytes. Their proliferation can promote follicular growth and development and thus promote ovulation, while apoptosis of granulosa cells may lead to follicular atresia. Pyruvate, as an important organic small molecule, is both a weak organic acid and has the characteristics of carboxylic acid, ketone and α-keto acid. After pyruvate enters the mitochondria, it is oxidized to generate acetyl-CoA, and then enters the tricarboxylic acid cycle, and finally is oxidized to carbon dioxide and water, completing the aerobic oxidation energy supply process of glucose. The tricarboxylic acid cycle is not only the intersection of catabolism and anabolism, but also has the basic function of oxidizing nutrients to support cell energy metabolism. Through pathway enrichment analysis, it was found that the tricarboxylic acid cycle is closely related to follicular development.

[0003] DLAT (dihydrolipoamide acetyltransferase) is the E2 subunit of the pyruvate dehydrogenase complex (PDHc). Studies by Golias and Patel et al. have shown that DLAT catalyzes the transfer of an acetyl group from dihydrolipoate to coenzyme A, converting pyruvate into acetyl-CoA and CO2, which is the only way for pyruvate to be converted into acetyl-CoA after entering the mitochondria. Goh et al. found that knocking down the DLAT gene would block the process of pyruvate conversion into acetyl-CoA, resulting in the accumulation of pyruvate in cells. Therefore, DLAT is indispensable in the tricarboxylic acid cycle. The research by Wang Ting et al. showed that the expression level of DLAT in breast cancer, prostate cancer and non-small cell lung cancer tissues was significantly higher than that in normal tissues. The research by Shan and Sun Mingming et al. pointed out that in a variety of tumor cells, DLAT can promote tumor cell growth by promoting the activation of the pentose phosphate pathway, and enhance its enzyme activity by acetylating 6-phosphogluconate dehydrogenase, thereby increasing nucleic acid synthesis and promoting the proliferation of lung cancer cells. However, there are relatively few studies on the effect of DLAT gene on follicular development at present. Summary of the Invention

[0004] In order to overcome the disadvantages and deficiencies of the prior art, the first object of the present invention is to provide the application of DLAT gene in promoting estrus in mammals.

[0005] The second object of the present invention is to provide the application of DLAT gene in promoting follicular development in mammals.

[0006] The third object of the present invention is to provide the application of the DLAT gene in the culture of ovarian granulosa cells.

[0007] The object of the present invention is achieved by the following technical solutions:

[0008] The application of the DLAT gene in the estrus of mammals is at least one of the following applications 1 and 2:

[0009] Application 1: The application of overexpressing the DLAT gene in promoting the estrus of mammals;

[0010] Application 2: The application of the overexpression vector of the DLAT gene in the preparation of products for promoting the estrus of mammals.

[0011] Further, the promotion of mammalian estrus is to shorten the estrus age of mammals.

[0012] Further, the product is any one of drugs, reagents, feeds, and feed additives.

[0013] Further, the dosage form of the drug or reagent is any one of oral dosage forms and injection dosage forms.

[0014] Further, the drug or reagent further comprises a pharmaceutically acceptable excipient or carrier.

[0015] Further, the mammal is any one of humans, mice, and pigs.

[0016] The application of the DLAT gene in follicular development is at least one of the following applications 3 and 4:

[0017] Application 3: The application of overexpressing the DLAT gene in promoting follicular development of mammals;

[0018] Application 4: The application of the overexpression vector of the DLAT gene in the preparation of products for promoting follicular development of mammals.

[0019] Further, the promotion of mammalian follicular development is to increase the number of corpora lutea and / or antral follicles.

[0020] Further, the product is any one of drugs and reagents.

[0021] Further, the drug or reagent further comprises a pharmaceutically acceptable excipient or carrier.

[0022] Further, the mammal is any one of humans, mice, and pigs.

[0023] The application of the DLAT gene in the culture of ovarian granulosa cells is at least one of the following applications 5 and 6:

[0024] Application 5: Application of overexpressing DLAT gene in in vitro culture of ovarian granulosa cells;

[0025] Application 6: Application of DLAT gene overexpression vector in the preparation of additives for in vitro culture of ovarian granulosa cells.

[0026] Furthermore, the Application 5 is as follows: Under in vitro conditions, overexpress the DLAT gene in ovarian granulosa cells to promote the proliferation of ovarian granulosa cells and inhibit the apoptosis of ovarian granulosa cells.

[0027] Furthermore, the in vitro culture additive is at least one of a proliferation promoter and an apoptosis inhibitor.

[0028] Furthermore, the ovarian granulosa cells are any one of human, mouse, and porcine ovarian granulosa cells.

[0029] Furthermore, the full-length genomic sequence of the human DLAT gene is as shown in the sequence with the NCBI accession number Gene ID: 1737, and the cDNA is as shown in the sequence with the NCBI accession number NM_001372041.1.

[0030] Furthermore, the full-length genomic sequence of the mouse DLAT gene is as shown in the sequence with the NCBI accession number Gene ID: 235339, the cDNA sequence is as shown in the sequence with the NCBI accession number 145614.4, and the CDS region sequence is as shown in the sequence with the NCBI accession number CCDS23168.1.

[0031] Furthermore, the basic plasmid used for the overexpression vector is pcDNA3.1.

[0032] Application of DLAT gene as a marker for ovarian follicle growth and development.

[0033] The verification results of the present invention are as follows:

[0034] 1. The relative expression level of DLAT in follicles with a diameter of >3 mm is significantly higher than that in follicles with a diameter of <3 mm.

[0035] 2. Treat granulosa cells with pyruvate and detect the relative expression level of DLAT by qRT-PCR. It is found that increasing pyruvate can significantly increase the relative expression level of DLAT.

[0036] 3. Design primers for the target gene DLAT: Search for the sequence of the target gene DLAT (NCBI Gene ID: 1737) on NCBI, determine the restriction enzyme sites (the restriction enzyme sites of the DLAT gene are BamHI and XbaI), use Primer Premier 5.0 software for primer design, specifically amplify, purify, and digest the target fragment by PCR, then ligate the pcDNA3.1 vector, and finally construct the overexpression vector pcDNA3.1-DLAT of the target gene DLAT. Subsequently, by transfecting the overexpression vector at different concentrations (100, 200, and 500 ng / mL) into ovarian granulosa cells and detecting the expression level of DLAT by qRT-PCR, it was found that the higher the concentration of the transfected overexpression vector, the better the transfection efficiency, and there were significant differences. Subsequently, 200 ng / mL was selected as the transfection concentration of pcDNA3.1-DLAT for subsequent research.

[0037] 4. Synthesize 3 pairs of small interfering fragments / controls for DLAT (si-DLAT / si-NC), screen and detect their interference efficiency. The results showed that after transfecting the gene interfering small fragments into porcine ovarian granulosa cells and using qRT-PCR, the si-DLAT2 small fragment with better interference effect was finally selected for subsequent experiments.

[0038] si-DLAT1: 5′-CCTGCTGGACCAAAGGGAA-3′;

[0039] si-DLAT2: 5′-TGGATGGAGCAGTTGGAGC-3′;

[0040] si-DLAT3: 5′-GTGCTTCAGAGGATAAACT-3′.

[0041] 5. Transfect pcDNA3.1-DLAT or si-DLAT2 into ovarian granulosa cells respectively, and use EdU and Annexin V-FITC to detect the effects of DLAT on the proliferation and apoptosis of ovarian granulosa cells. The results showed that the proliferation rate of the pcDNA3.1-DLAT group was significantly higher than that of the control group pcDNA3.1, and the proliferation rate of the si-DLAT2 group was significantly lower than that of the control group si-NC. The apoptosis detection results showed that the apoptosis rate (early apoptosis + late apoptosis) of the pcDNA3.1-DLAT group was significantly lower than that of the control group pcDNA3.1, and the apoptosis rate of the si-DLAT2 group was significantly higher than that of the control group si-NC.

[0042] 6. Transfect pcDNA3.1-DLAT or si-DLAT2 into ovarian granulosa cells respectively. After 24 hours, extract RNA to detect the mRNA levels of key genes in the proliferation pathway. The mRNA levels of key genes in the proliferation pathway are upregulated after overexpressing DLAT, and downregulated after interfering with DLAT. Detect the mRNA levels of key genes in the apoptosis pathway. The mRNA levels of key genes in the apoptosis pathway are downregulated after overexpressing DLAT, and upregulated after interfering with DLAT. Extract proteins to detect the protein levels of key genes in the proliferation pathway. The protein levels of key genes in the proliferation pathway are upregulated after overexpressing DLAT, and downregulated after interfering with DLAT. Detect the protein levels of key genes in the apoptosis pathway. The protein levels of key genes in the apoptosis pathway are downregulated after overexpressing DLAT, and upregulated after interfering with DLAT.

[0043] 7. Extract granulosa cell proteins and perform immunoprecipitation on DLAT using CoIP to pull down proteins related to the cell apoptosis pathway. The results show that DLAT protein binds to the proteins of CASP3 and CASP9.

[0044] 8. Isolate the follicles from the ovaries of healthy sows and infect the follicles with DLAT lentivirus. Observe the status of the follicles on the first, third, and fifth days after infection and record them. Then extract RNA and proteins and verify them using qRT-PCR and Western Blot respectively. The results show that overexpressing DLAT increases the blood vessels on the surface of the follicles. After interfering with DLAT expression, the increase in blood vessels on the surface of the follicles is reduced. Overexpressing DLAT upregulates the mRNA and protein levels of genes related to the proliferation pathway in the follicles, and interfering with DLAT expression downregulates the mRNA and protein levels of genes related to the proliferation pathway in the follicles. Overexpressing DLAT downregulates the mRNA and protein levels of genes related to the apoptosis pathway in the follicles, and interfering with DLAT expression upregulates the mRNA and protein levels of genes related to the apoptosis pathway in the follicles.

[0045] 9. Mice were intraperitoneally injected with DLAT overexpression and interfering lentiviruses, the estrus age of the mice was recorded, and ovarian samples were collected 3 weeks later to extract RNA and proteins. The results showed that overexpression of DLAT significantly upregulated the mRNA and protein levels of key genes in the proliferation pathway, while interference with DLAT significantly downregulated the mRNA and protein levels of key genes in the proliferation pathway. Overexpression of DLAT downregulated the mRNA and protein levels of genes related to the apoptosis pathway in follicles, and interference with DLAT expression upregulated the mRNA and protein levels of genes related to the apoptosis pathway in follicles. Mouse ovarian proteins were extracted, and DLAT was immunoprecipitated using CoIP to pull down proteins related to the cell apoptosis pathway. The results also showed that DLAT protein bound to the proteins of CASP3 and CASP9. In summary, DLAT can promote the proliferation of ovarian granulosa cells, inhibit apoptosis, and thus promote follicle development.

[0046] The present invention has the following advantages and effects compared with the prior art:

[0047] 1. The present invention used two types of follicles with diameters >3 mm and <3 mm as experimental materials. Through qRT-PCR, it was found that in sow follicles with a diameter <3 mm, the expression level of the DLAT gene was significantly lower than that in follicles with a diameter >3 mm.

[0048] 2. The present invention took DLAT as the research object and used molecular and cell biology methods to study its application in ovarian granulosa cells: by increasing the level of pyruvate, it was found that increasing pyruvate could promote the transcription of the DLAT gene. By overexpressing or interfering with DLAT, it was found that DLAT could promote the proliferation of ovarian granulosa cells and inhibit apoptosis. By infecting porcine follicles with DLAT lentivirus, it was found that DLAT could promote follicle development. By injecting DLAT lentivirus, it was found that DLAT could promote the development of ovarian follicles in mice and promote estrus. In addition, it was also found that DLAT protein bound to the proteins of CASP3 and CASP9 in both granulosa cells and mouse ovaries. It has good application value for studying the mechanism of ovarian follicle development, etc.

[0049] 3. The technical solution of the present invention is well-designed and the results are reliable. Description of the Drawings

[0050] Figure 1 It is a graph of the relative expression level of DLAT in follicles of different sizes.

[0051] Figure 2 It is a graph of the effect of pyruvate on the relative expression level of DLAT.

[0052] Figure 3 It is an electrophoresis diagram of double digestion of the pcDNA3.1-DLAT recombinant plasmid.

[0053] Figure 4It is a diagram for detecting the efficiency of the overexpression vector pcDNA3.1-DLAT.

[0054] Figure 5 It is a diagram for detecting the efficiency of the interference fragment si-DLAT.

[0055] Figure 6 It is a diagram showing the effect of overexpressing or interfering with DLAT on the proliferation of ovarian granulosa cells; among them, a is the effect of overexpressing DLAT on the proliferation of ovarian granulosa cells detected by the EdU method; b is the effect of interfering with DLAT on the proliferation of ovarian granulosa cells detected by the EdU method; c is the effect of overexpressing or interfering with DLAT on the mRNA level of genes related to the granulosa cell proliferation pathway; d is the effect of overexpressing or interfering with DLAT on the protein level of genes related to the granulosa cell proliferation pathway.

[0056] Figure 7 It is a diagram showing the effect of overexpressing or interfering with DLAT on the apoptosis of granulosa cells; among them, a is the effect of overexpressing DLAT on the apoptosis of granulosa cells; b is to detect the effect of interfering with DLAT on the apoptosis of ovarian granulosa cells; c is the effect of overexpressing or interfering with DLAT on the mRNA level of genes related to the granulosa cell apoptosis pathway; d is the effect of overexpressing or interfering with DLAT on the protein level of genes related to the granulosa cell apoptosis pathway.

[0057] Figure 8 It is a CoIP result diagram of the interaction between DLAT protein and other proteins in granulosa cells.

[0058] Figure 9 It is a diagram showing the effect of DLAT on porcine follicular development; among them, a is the follicular status on the first, third, and fifth days after porcine follicles are treated with DLAT lentivirus; b is the effect of overexpressing or interfering with DLAT lentivirus on the mRNA level of genes related to the granulosa cell proliferation and apoptosis pathway; c is the effect of overexpressing or interfering with DLAT lentivirus on the protein level of genes related to the granulosa cell proliferation and apoptosis pathway.

[0059] Figure 10 It is a diagram for detecting the efficiency of injecting DLAT lentivirus into mice.

[0060] Figure 11 It is a diagram showing the effect of DLAT on mouse follicular development; among them, a is the statistical result of the estrus age of mice after lentivirus injection; b is the HE staining diagram of the ovaries of mice after lentivirus treatment.

[0061] Figure 12 It is a diagram showing the effect of overexpressing or interfering with DLAT lentivirus on the mRNA and protein levels of genes related to the ovarian proliferation pathway in mice.

[0062] Figure 13 It is a Tunel staining diagram of mouse ovaries.

[0063] Figure 14 It is a graph showing the effects of overexpressing or interfering with DLAT lentivirus on the mRNA and protein levels of genes related to the apoptosis pathway in mouse ovaries.

[0064] Figure 15 It is a CoIP result graph of the interaction between DLAT protein and other proteins in mouse ovaries. Detailed implementation methods

[0065] The present invention will be further described in detail below in conjunction with embodiments and the accompanying drawings, but the implementation manners of the present invention are not limited thereto. For the experimental methods without specific conditions noted in the following embodiments, they are usually carried out according to conventional conditions. Unless otherwise specified, the reagents and raw materials used in the present invention can be obtained commercially.

[0066] In the present invention, statistical methods are applied to analyze the results of 3 independent experiments in each embodiment, calculate "mean ± standard deviation" respectively, and use one-way ANOVA for significant difference analysis (in the figure, "*" indicates P < 0.05, and "**" indicates P < 0.01).

[0067] Example 1: Detection of relative expression levels of DLAT in follicles of different sizes

[0068] (1) RNA extraction

[0069] ① Extract two types of follicles with diameters < 3 mm and > 3 mm from the ovaries of sows (healthy commercial sows from Guangzhou Kongwangji Slaughterhouse). Thoroughly homogenize the samples after adding TRIzol in proportion, let them stand on ice for 10 min, centrifuge at 4°C and 12,000 rpm for 5 min;

[0070] ② Transfer the supernatant to a new 1.5 mL sterilized centrifuge tube, add 200 μL of chloroform, shake and mix well for 30 s, let it stand on ice for 15 min, centrifuge at 4°C and 12,000 rpm for 15 min;

[0071] ③ Transfer the upper aqueous phase to a new 1.5 mL sterilized centrifuge tube, add 500 μL of isopropanol, gently mix, let it stand on ice for 15 min, centrifuge at 4°C and 12,000 rpm for 15 min, and discard the supernatant;

[0072] ④ Add 1 mL of pre-cooled 75% ethanol (ethanol:DEPC water = 3:1), resuspend and wash the RNA precipitate, centrifuge at 4°C and 12,000 rpm for 5 min;

[0073] ⑤ Discard the supernatant, leave the RNA precipitate, dry for 5 min, and then add 30 μL of DEPC water to resuspend and mix the RNA precipitate evenly. Use a UV spectrophotometer to detect the OD 260 / OD 280 ratio for quality detection, and store the qualified RNA in a -80°C refrigerator.

[0074] (2) RNA reverse transcription:

[0075] Configure the system according to the PrimeScript RT Master Mix instruction manual. The reaction system is shown in Table 1.

[0076] Table 1 RNA reverse transcription system

[0077]

[0078] Put the configured system into a PCR instrument and perform the reaction at 37°C for 15 min and 85°C for 5 s. The reverse-transcribed cDNA is placed in a -20°C refrigerator for subsequent experiments.

[0079] The results are as Figure 1 shown. The relative expression level of DLAT in follicles with a diameter > 3 mm is significantly higher than that in follicles < 3 mm.

[0080] Example 2: Effects of overexpressing or interfering with DLAT on ovarian granulosa cells

[0081] 1. Culture of ovarian granulosa cells

[0082] (1) Cell resuscitation: Wait for the water bath to heat up to 37°C. Place the cryopreserved KGN cells in the water bath, heat for 1 min, and shake the cryopreservation tube to accelerate cell thawing. Add complete medium to a centrifuge tube, add the thawed cell suspension to the centrifuge tube, mix well, and centrifuge at 1,000 rpm for 5 min. Pour off the supernatant, add 5 ml of complete medium to the precipitate in the centrifuge tube for resuspension, transfer the cell suspension to a small culture flask, mix the suspension well, and place it in an incubator for culture.

[0083] (2) Cell passage: Pour off the medium in the flask, wash the culture flask twice with PBS, add 3 ml of trypsin, and perform digestion in the incubator for 5 min. Use an equal volume of complete medium to terminate the digestion. Place the cell suspension in a centrifuge tube and centrifuge at 1,000 rpm for 5 min. Pour off the supernatant, wash twice with PBS again, pour off the supernatant, resuspend the cells with 2 ml of complete medium, transfer the suspension to a large culture flask, mix the suspension well, and place it in a 37°C incubator for culture.

[0084] 2. Treat ovarian granulosa cells with pyruvate

[0085] (1) When the cell density in the flask grows to about 80%, pour out the culture medium in the flask. After washing the culture flask twice with PBS, add 3 ml of trypsin and incubate in the incubator for 5 minutes for digestion. Add an equal volume of complete culture medium to end the digestion. Transfer the cell suspension into a centrifuge tube and centrifuge at a speed of 1,000 rpm for 5 minutes. Pour out the supernatant, wash twice with PBS, pour out the supernatant again, and resuspend the cells with 2 ml of complete culture medium;

[0086] (2) Divide the cells into two culture flasks and culture for 24 hours first. Then, pour out the culture medium in the flask. After washing the culture flask twice with PBS, treat one flask with 1 mM pyruvate, and one group is the blank group. After culturing for 24 hours, conduct subsequent experiments.

[0087] 3. Construct the overexpression vector and interference vector of the DLAT gene

[0088] (1) Search for the human DLAT gene (GeneID: 1737, NM_001372041.1) on NCBI and use Primer Premier 5.0 software for primer design. Extract the total RNA of KGN granulosa cells, and then reverse transcribe it into cDNA using a kit as a template for amplification. Purify and recover the amplified fragment, ligate it to the pMD18T vector (Takara), transform it. After detecting the bacterial liquid and performing sequencing identification correctly, extract the ordinary plasmid and name it T-DLAT. The primer sequences are as follows:

[0089] F: 5′-CCCGCATCAGAAGGTTCCAT-3′;

[0090] R: 5′-TCAGTGTGACCTGGGAGAGT-3′.

[0091] (2) Add BamHI and EcoRI restriction enzyme site sequences to the upstream and downstream primers respectively. Use the ordinary plasmid T-DLAT as a template for PCR amplification; recover and purify the target fragment, double-digest the pcDNA3.1 vector (Invitrogen, catalog number V79020) and the target fragment, ligate pcDNA3.1, transform, screen and sequence for identification. After correct identification, extract the endotoxin-free plasmid (the endotoxin-free plasmid small-scale extraction kit is purchased from Magen, USA) and name it pcDNA3.1-DLAT. The DLAT gene restriction enzyme primer sequences are as follows:

[0092] F: 5′-CGGGATCCCCCGCATCAGAAGGTTCCAT-3′;

[0093] R: 5′-CGGAATTCTCAGTGTGACCTGGGAGAGT-3′.

[0094] (3) Entrust Guangzhou Ribobio Co., Ltd. to synthesize 3 pairs of small interfering fragments against DLAT / control (si-DLAT / si-NC):

[0095] si-DLAT1: 5′-CCTGCTGGACCAAAGGGAA-3′;

[0096] si-DLAT2: 5′-TGGATGGAGCAGTTGGAGC-3′;

[0097] si-DLAT3: 5′-GTGCTTCAGAGGATAAACT-3′.

[0098] 4. Seeding and transfection of ovarian granulosa cells

[0099] (1) First, it is necessary to seed the cells. When the cell density in the flask reaches about 80%, pour out the culture medium in the flask. After washing the culture flask twice with PBS, add 3 ml of trypsin and incubate in the incubator for 5 minutes for digestion. Add an equal volume of complete culture medium to terminate the digestion. Transfer the cell suspension into a centrifuge tube and centrifuge at a speed of 1,000 rpm for 5 minutes. Pour out the supernatant, wash twice with PBS, pour out the supernatant again, and resuspend the cells with 3 ml of complete culture medium;

[0100] (2) According to different specifications of cell culture plates, add the corresponding amount of cell suspension, gently mix, and place in a 37°C incubator for culture;

[0101] (3) When the cell density in the plate reaches about 80%, pour out the culture medium in the flask, wash the culture flask twice with PBS, add incomplete culture medium (without double antibiotics), and finally perform the above-mentioned recombinant vector transfection or drug treatment;

[0102] (4) Refer to the instruction manual of 3000 Transfection Reagent to prepare the transfection mixture;

[0103] (5) Add the prepared transfection mixture to the plate according to the ratio, mix well, place in the incubator, and perform subsequent experiments.

[0104] 5. qRT-PCR

[0105] The qRT-PCR detection in the present invention uses qPCR SYBR Green Master Mix kit. The comparative Ct value method is used in the experiment to detect the content of sample genes. The specific calculation formula is as follows:

[0106] Relative gene expression = 2 -{〈﹙实验组目的基因Ct值﹚-﹙实验组内参基因Ct值﹚〉-〈﹙对照组目的基因Ct值﹚-﹙对照组内参基因Ct值﹚〉}

[0107] GAPDH was used as an internal reference for detecting genes. The qRT-PCR primers used in this invention are as follows:

[0108] qRT-PCR-DLAT Forward: 5′-AGCAGCACGACTAGAGGGTATGG-3′;

[0109] Reverse: 5′-TACCAGCGGCGGTTAGGAGAC-3′;

[0110] qRT-PCR-GAPDH Forward: 5′-GGACTCATGACCACGGTCCAT-3′;

[0111] Reverse: 5′-TCAGATCCACAACCGACACGT-3′.

[0112] 6. Protein extraction

[0113] Wash the cells with pre-cooled PBS buffer, add an appropriate amount of protein lysate (containing 1% protease inhibitor), incubate on a shaker at 4°C for 15 min, blow the cells in the wells to fully lyse them, repeat the addition into the same tube, centrifuge at 12,000 rpm at 4°C for 10 min, and transfer the supernatant to a new centrifuge tube.

[0114] 7. Detection of ovarian granulosa cell proliferation

[0115] In this invention, the EdU method was used to detect cell proliferation. Referring to the instruction manual of the Cell-Light TM EdU Apollo 567 In vitro Kit detection kit, the specific operation steps are as follows:

[0116] (1) Dilute the EdU solution with complete medium to a final concentration of 50 μM, add 150 μL of the EdU dilution to each well, place the culture plate in the incubator and incubate for 2 h, discard the medium, and wash the cells 2 times with PBS solution;

[0117] (2) Add 150 μL of fixative (PBS containing 80% acetone) to each well, incubate at room temperature for 30 min, discard the fixative, and wash 2 times with PBS;

[0118] (3) Add 150 μL of PBS containing 0.5% Triton X to each well to permeabilize the cells for 10 min, and wash the cells 3 times with PBS;

[0119] (4) Prepare 1× Apollo staining reaction solution according to the instruction manual, add 150 μL to each well, incubate in the dark at room temperature for 30 min, aspirate and discard the reaction solution, and wash 3 times with PBS;

[0120] (5) Prepare DAPI staining solution, add 150 μL to each well, incubate at room temperature in the dark for 30 min, aspirate the reaction solution, and wash 3 times with PBS;

[0121] (6) Add 150 μL of PBS solution to each well to avoid drying at the bottom of the wells. Take pictures using a fluorescence microscope. The cells stained blue in the pictures are the cells on the cell culture plate before fixation, and the cells stained red are the newly proliferated cells on the cell culture plate within 30 min of incubation. Finally, use ImageJ to count the number of granulosa cells and analyze the proliferation rate of granulosa cells.

[0122] 8. Detection of apoptosis of ovarian granulosa cells

[0123] This invention uses Annexin V-FITC technology to detect cell apoptosis. Referring to the instruction manual of the Annexin V-FITC Apoptosis Detection Kit from BioVision, the specific operation steps are as follows:

[0124] (1) Inoculate porcine ovarian granulosa cells into a 6-well plate, culture the cells until the confluence reaches 50 - 80%, and wash the cells with PBS solution;

[0125] (2) Digest the cells with trypsin without EDTA (ethylenediaminetetraacetic acid), and wash the cells with 2 mL of PBS solution;

[0126] (3) Take 0.5 mL of cell suspension (about 5×10 5 cells), and add 500 μL of 1×Binding Buffer;

[0127] (4) Add 5 μL of Annexin V-FITC and 5 μL of PI (Propidium Iodide) at room temperature, and incubate at room temperature in the dark for 5 min;

[0128] (5) Immediately detect and analyze using a flow cytometer.

[0129] To explore the effect of pyruvate on the DLAT gene, porcine ovarian granulosa cells were treated with pyruvate. The experiment was divided into two groups: pyruvate group and blank group. The relative expression level of DLAT was detected by qRT-PCR. The results showed that increasing pyruvate could significantly increase the relative expression level of DLAT ( Figure 2 ).

[0130] The described ovarian follicles are derived from healthy commercial sows in Kong Wangji Slaughterhouse, Guangzhou.

[0131] Primers for the designed target gene DLAT: Search for the sequence of the target gene DLAT (NCBI Gene ID: 1737) on NCBI, perform specific amplification using NCBI. After successful sequencing, determine the restriction enzyme sites, and use Primer Premier 5.0 software for primer design. Specifically amplify, purify, and digest the target fragment by PCR, and then ligate it with the pcDNA3.1 expression vector to construct the overexpression vector pcDNA3.1-DLAT of the target gene DLAT( Figure 3 ). Transfect the overexpression vector at different concentrations (100, 200, and 500 ng / mL) into ovarian granulosa cells, and use qRT-PCR to detect the expression level of DLAT. The results are as Figure 4 shown. The higher the concentration of the transfected pcDNA3.1-DLAT overexpression vector, the better the transfection efficiency, and the difference is significant. The subsequent study selected 200 ng / mL as the transfection concentration of pcDNA3.1-DLAT.

[0132] Synthesize 3 pairs of small interfering fragments / controls for DLAT (si-DLAT / si-NC), screen and detect their interference efficiency. The results are as Figure 4 shown. Transfect the gene interfering small fragments into ovarian granulosa cells, and finally screen the si-DLAT2 small fragment with better interference effect for subsequent experiments by qRT-PCR.

[0133] Transfect the above control groups pcDNA3.1 and pcDNA3.1-DLAT, as well as the control groups si-NC and si-DLAT2 into ovarian granulosa cells respectively, and use the EdU method and Annexin V-FITC method to detect the effects of DLAT on the proliferation and apoptosis of ovarian granulosa cells. The results show that the proliferation rate of the pcDNA3.1-DLAT group is significantly higher than that of the control group pcDNA3.1( Figure 6 in a), and the proliferation rate of the si-DLAT2 group is significantly lower than that of the control group si-NC( Figure 6 in b). Another part of the results shows that the apoptosis rate (early apoptosis + late apoptosis) of the pcDNA3.1-DLAT group is significantly lower than that of the control group pcDNA3.1( Figure 7 in a), and the apoptosis rate of the si-DLAT2 group is significantly higher than that of the control group si-NC( Figure 7 in b).

[0134] Extract granulosa cell proteins, perform immunoprecipitation on DLAT using CoIP, and pull down the proteins related to the cell apoptosis pathway. The results show that the DLAT protein binds to the proteins of CASP3 and CASP9( Figure 8 ).

[0135] Example 3: Effects of overexpression or interference of DLAT on follicular development

[0136] 1. Lentivirus construction

[0137] Entrust Guangzhou Dongze Biotechnology Co., Ltd. to package the overexpressing lentivirus and knockdown lentivirus of DLAT. Among them, the overexpressing lentivirus is obtained by constructing the DLAT gene (NM_145614.4) into the pLVX-C-FLAG-PGK-Puro plasmid through the XhoI + MluI restriction enzyme sites, importing it into 293T cells, and producing a high-titer lentivirus containing the target gene, named rLV-mDLAT; the knockdown lentivirus is obtained by constructing the shRNA for knocking down DLAT (target sequence: 5'-TGGATGGAGCAGTTGGAGC-3') into the pLVX-shRNA1 plasmid through the BamHI + EcoRI restriction enzyme sites, importing it into 293T cells, and producing a high-titer lentivirus containing the target gene, named rLV-shRNA. Among them, the inserted fragment sequences are as follows:

[0138]

[0139] 2. Isolation and culture of ovarian follicles

[0140] (1) Add 1% penicillin-streptomycin to PBS and DMEM / F12 medium in advance, and wash the collected sow ovaries with PBS until there is no bloody water;

[0141] (2) Under the laminar flow hood, use a surgical blade to isolate follicles with a diameter of 3 - 5 mm. Wash the isolated follicles twice with PBS, then wash them twice with DMEM / F12 medium. Add 500 μL of DMEM / F12 medium to a 24-well plate, and place the follicles into the 24-well plate (1 follicle / well), and culture them under the conditions of 38.5 °C and 5% CO2.

[0142] (3) After 24 h, randomly divide them into four groups: rLV-puro group, rLV-mDLAT group, rLV-shRNA group, and rLV-shRNA-mDLAT group. Treat the follicles with lentivirus (titer is 10 7 ), and take pictures at 1, 3, and 5 d after treatment.

[0143] 3. Other experiments refer to Example 1 and Example 2.

[0144] Infect sow follicles with lentivirus, and detect the expression levels of genes related to the proliferation and apoptosis pathways by qRT-PCR and Western Blot. The results show that DLAT can promote the expression of genes in the proliferation pathway in follicles and inhibit the expression of genes in the apoptosis pathway, thereby promoting follicle development ( Figure 9 ).

[0145] Example 4: Effects of overexpressing or interfering with DLAT on mouse estrus

[0146] 1. Feeding of mice and lentivirus treatment

[0147] Select 32 21-day-old C57BL / 6J female mice, randomly divide them into four groups: rLV-puro group, rLV-mDLAT group, rLV-shRNA group, and rLV-shRNA-mDLAT group, with 8 replicates in each group. After adaptively feeding them in the Experimental Animal Center of South China Agricultural University for 1 week, inject the lentiviral vector into the mice by intraperitoneal injection. The injection titer is 1×10 7 , once a week for three consecutive weeks. Weigh the mice every day and observe the estrus situation of the mice and record it. Sacrifice the mice at 42 days old and take the mouse ovaries.

[0148] 2. HE staining

[0149] The present invention uses hematoxylin-eosin staining (HE) to detect the follicular development, and the specific experimental steps are referred to the official website of Wuhan Saiweier Biotechnology Co., Ltd.

[0150] 3. Detection of ovarian follicle apoptosis by Tunel

[0151] The present invention uses paraffin section fluorescence Tunel to detect the apoptosis of ovarian follicles, and the specific experimental steps are referred to the official website of Wuhan Saiweier Biotechnology Co., Ltd.

[0152] 4. Other experiments refer to Examples 1, 2, and 3.

[0153] DLAT lentivirus injection was performed on mice ( Figure 10 ). The HE staining results showed that overexpression of DLAT increased the number of corpora lutea ( Figure 11 ). Tunel detection found that overexpression of DLAT reduced the apoptosis of ovarian granulosa cells ( Figure 13 ), while interfering with DLAT gave the opposite results. The qRT-PCR and Western Blot results showed that DLAT could promote the expression of genes in the proliferation pathway in the ovary and inhibit the expression of genes in the apoptosis pathway, thereby promoting estrus ( Figure 12 、 Figure 14 ).

[0154] Mouse ovarian proteins were extracted, and CoIP was used to immunoprecipitate DLAT to pull down proteins related to the cell apoptosis pathway. The results showed that DLAT protein was combined with the proteins of CASP3 and CASP9 ( Figure 15 ).

[0155] In summary, DLAT can promote the proliferation of ovarian granulosa cells, and DLAT protein inhibits cell apoptosis by binding to the proteins of CASP3 and CASP9, thereby promoting follicular development and promoting estrus in mice.

[0156] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. Use of the DLAT gene in mammalian estrus, characterized in that: For at least one of the following applications 1 and 2: Application 1: Application of overexpressing the DLAT gene in promoting estrus in mammals; Application 2: Application of a DLAT gene overexpression vector in the preparation of a product for promoting estrus in mammals.

2. The application according to claim 1, wherein: The promotion of estrus in mammals is to shorten the estrus age of mammals; The mammal is any one of human, mouse, and pig.

3. The application according to claim 1, wherein: The product is any one of a drug, a reagent, a feed, and a feed additive; The dosage form of the drug or reagent is any one of an oral dosage form and an injection dosage form; The drug or reagent further comprises a pharmaceutically acceptable excipient or carrier.

4. Application of the DLAT gene in follicular development, for at least one of the following applications 3 and 4: Application 3: Application of overexpressing the DLAT gene in promoting follicular development in mammals; Application 4: Application of a DLAT gene overexpression vector in the preparation of a product for promoting follicular development in mammals.

5. The application according to claim 4, wherein: The promotion of follicular development in mammals is to increase the number of corpora lutea and / or antral follicles; The mammal is any one of human, mouse, and pig.

6. The application according to claim 4, wherein: The product is any one of a drug and a reagent; The drug or reagent further comprises a pharmaceutically acceptable excipient or carrier.

7. Application of the DLAT gene in ovarian granulosa cell culture, for at least one of the following applications 5 and 6: Application 5: Application of overexpressing the DLAT gene in in vitro culture of ovarian granulosa cells; Application 6: Application of a DLAT gene overexpression vector in the preparation of an additive for in vitro culture of ovarian granulosa cells.

8. The application according to claim 7, wherein: The Application 5 is: under an in vitro environment, overexpressing the DLAT gene in ovarian granulosa cells to promote the proliferation of ovarian granulosa cells and inhibit the apoptosis of ovarian granulosa cells; The in vitro culture additive is at least one of a proliferation promoter and an apoptosis inhibitor; The ovarian granulosa cells are any one of human, mouse, and pig ovarian granulosa cells.

9. The application according to claim 8, wherein: The basic plasmid used for the overexpression vector is pcDNA3.

1.

10. Application of the DLAT gene as a marker for ovarian follicle growth and development.

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