Application of transcription factor E2F5 for regulating and controlling chicken fat formation

Abstract

The invention discloses application of a transcription factor E2F5 for regulating and controlling chicken fat formation, and relates to the field of animal molecular heredity and developmental biology. Differentiation of chicken preadipocytes is inhibited through overexpression of the transcription factor E2F5, differentiation of the chicken preadipocytes is promoted through interference of the E2F5, and the transcription factor KLF7 plays a regulating role by regulating the enhancer activity of the transcription factor E2F5.

Description

Technical Field:

[0001] The present invention relates to the fields of animal molecular genetics and developmental biology, and particularly to the application of a transcription factor E2F5 for regulating chicken fat formation. Background Art:

[0002] Diseases such as hypertension, cardiovascular diseases, non-alcoholic fatty liver, type II diabetes, etc. [1] caused by obesity bring many troubles to human life and production. In the broiler industry, the impact of fat deposition in chickens on feed conversion rate, egg production, and hatching ratio cannot be ignored. From the aspects of chicken's natural hyperglycemia, insulin resistance, hepatic fatty acid synthesis, and reproductive system, chickens are ideal models for studying fat biology. Especially in adipose tissue, the hyperglycemia and low sensitivity to exogenous insulin in chickens make them relevant models for studying human obesity, insulin resistance, and type II diabetes. Therefore, in-depth understanding of the molecular mechanism of chicken adipogenesis has important theoretical significance and practical application value for improving meat quality and breeding low-fat poultry lines.

[0003] Large-scale analysis of human fat gene expression using microarray technology found that the newly identified genes with the highest upregulation related to fatty acid metabolism include the E2F5 transcription factor. The E2F family participates in the regulation of cellular functions during the adipogenic process of preadipocytes, and E2F5 is also expressed in human preadipocytes. Therefore, it is urgent to clarify the role of E2F5 in adipogenesis, screen a regulatory substance specific to E2F5, and clarify the regulatory mechanism therein, so as to develop new drug targets to effectively inhibit excessive fat accumulation in broilers and the occurrence of human obesity. At the same time, patients sensitive to drugs or treatments targeting E2F5 can be screened based on this regulatory substance or regulatory mechanism for precise treatment.

[0004] Summary of the Invention: The purpose of the present invention is the application of a transcription factor E2F5 for regulating chicken fat formation to solve the problems existing in the above-mentioned prior art.

[0005] To achieve the above purpose, the present invention provides the following solution:

[0006] The use of a transcription factor E2F5 for regulating the differentiation of chicken preadipocytes. The regulation of the differentiation of chicken preadipocytes refers to inhibiting the differentiation of chicken preadipocytes by overexpressing the transcription factor E2F5. The transcription factor KLF7 plays a regulatory role by regulating the enhancer activity of the transcription factor E2F5. The sequence of the transcription factor E2F5 containing enhancer activity is shown as SEQ ID No.1, and the sequence where the transcription factor KLF7 binds to the enhancer region of E2F5 is shown as SEQ ID No.2.

[0007] A transcription factor E2F5 for regulating chicken fat formation, and the coding gene sequence of the transcription factor E2F5:

[0008] Construct a recombinant plasmid pCMV-HA-E2F5 and transfect chicken preadipocytes. The amino acid sequence of the recombinant plasmid pCMV-HA-E2F5 is shown in SEQ ID No. 4.

[0009] An overexpression plasmid of chicken E2F5 gene is constructed by recombining the CDS sequence of chicken E2F5 gene and the pCMV-HA eukaryotic expression vector. The construction method steps are as follows:

[0010] (1) Design of primers for amplifying the CDS region of chicken E2F5 gene and PCR amplification

[0011] Forward primer:

[0012] Reverse primer:

[0013] (2) Recovery of E2F5 gene DNA fragment

[0014] (3) Double digestion of pCMV-HA eukaryotic expression vector

[0015] (4) Ligation and transformation of the PCR amplification product of the CDS region of chicken E2F5 gene and the linearized pCMV-HA eukaryotic expression vector

[0016] (5) Small-scale extraction of E2F5 gene plasmid DNA

[0017] (6) Identification of pCMV-HA-E2F5 eukaryotic expression vector

[0018] The present invention discloses the following technical effects:

[0019] The present invention discloses the application of a transcription factor E2F5 for regulating the formation of chicken preadipocytes; the present invention relates to a method for regulating the expression of E2F5 gene, which includes using a substance specifically regulating the expression of E2F5. The present invention further reveals the mechanism of action of transcription factor E2F5 in the formation of chicken preadipose tissue, thereby affecting the differentiation of chicken preadipocytes. The present invention provides a new use of transcription factor E2F5 in regulating chicken fat formation. The research results contribute to improving the molecular regulation network of adipose tissue development and provide important references for in-depth study of the function of E2F5 in adipose tissue development, and provide new ideas for the research of other members of the KLFs family. Approximately 60% of chicken genes are highly homologous to human genes. Chicken is a potential model for studying human obesity and obesity-related diseases. This study also provides a reference for further understanding the molecular mechanisms of human fat formation and obesity occurrence, and is of great significance for preventing obesity. Description of the drawings:

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0021] Figure 1 Schematic diagram of the prediction of the promoter site of the chicken E2F5 gene and the construction strategy of the promoter reporter gene vector, where (A) is the diagram of the predicted E2F5 promoter position by Promoter 2.0, and (B) is the diagram of the construction strategy of the E2F5 gene promoter reporter gene vector;

[0022] Figure 2 It is the diagram of verifying the enhancer activity of the +22661 / +22900 region by transfecting pGL3-Promoter-E2F5;

[0023] Figure 3 It is the diagram of double digestion identification of the recombinant plasmids P1, P2, P3, and P4;

[0024] Figure 4 It is the diagram of the promoter activity analysis of the chicken E2F5 gene;

[0025] Figure 5 It is the diagram of the enhancer activity analysis of chicken E2F5(+22661 / +22900);

[0026] Figure 6 It is the diagram of the analysis of the binding site of KLF7 to the 3′ flanking region of the E2F5 gene. Among them, (A) is the structural diagram of the binding site deletion mutant vector, (B) is the diagram of the change in luciferase activity after the binding site deletion mutation after overexpressing KLF7 in the immortalized chicken preadipocyte line (Immortalized chicken preadipocyte, ICP1), and (C) is the diagram of the change in luciferase activity after the binding site deletion mutation after overexpressing KLF7 in DF-1 cells;

[0027] Figure 7 It is the diagram of the analysis of the effect of overexpressing KLF7 on the endogenous expression of the E2F5 gene. Among them, (A) is the analysis diagram of the overexpression effect of the eukaryotic expression vector pCMV-Myc-KLF7, and (B) is the grayscale value quantification analysis diagram of the overexpression effect;

[0028] Figure 8It is a graph showing the lipid droplet deposition and the expression levels of adipogenic differentiation-related genes and E2F5 during the differentiation of ICP1. Among them, (A) is the Oil Red O staining identification graph of ICP1 cells induced to differentiate for 0, 48, and 96 h by oleic acid, (B) is the verification graph of the absorbance value measured at 510 nm for the extracted dye, (C) is the result graph of the expression levels of adipogenic differentiation-related genes PPARγ, FABP4, FAS, and GPDH analyzed by qRT-PCR, and (D) is the result graph of the expression level of E2F5 analyzed by qRT-PCR;

[0029] Figure 9 It is a graph of double digestion identification of the pCMV-HA-E2F5 vector and verification of the overexpression effect. Among them, (A) is the double digestion identification graph of the pCMV-HA-E2F5 vector, and (B) is the verification graph of the overexpression effect of the double digestion of the pCMV-HA-E2F5 vector;

[0030] Figure 10 It is a graph analyzing the effect of overexpressing E2F5 on the differentiation of the ICP1 cell line. Among them, (A) is the verification graph of Oil Red O staining after overexpressing E2F1, (B) is the extraction colorimetric analysis graph after Oil Red O staining after overexpressing E2F1, and (C) is the graph analyzing the effect of overexpressing E2F5 on the differentiation of the ICP1 cell line detected by BODIPY 493 / 503 staining;

[0031] Figure 11 It is a graph analyzing the effect of overexpressing E2F5 on the expression of adipogenic differentiation-related genes. Among them

[0032] (A) is a graph analyzing the expression (mean ± standard deviation) of adipogenic differentiation-related genes in ICP1 cells after overexpressing E2F5 detected by qRT-PCR with NONO as the internal reference,

[0033] (B) is a graph analyzing the expression of adipogenic differentiation-related genes in ICP1 cells after overexpressing E2F5 detected by Western Blot with β-actin as the internal reference;

[0034] (C) is a graph of quantitative analysis (mean ± standard deviation) of the western blot protein level;

[0035] Figure 12 It is a graph verifying the interference effect of sh-1, sh-2, and sh-3 on the E2F5 gene by qRT-PCR;

[0036] Figure 13It is an analysis diagram of the effect of interfering with E2F5 on the differentiation of ICP1 cell line. Among them, (A) is the verification diagram of Oil Red O staining after interfering with E2F1, (B) is the extraction colorimetric analysis diagram after Oil Red O staining after interfering with E2F1, and (C) is the analysis diagram of the effect of BODIPY 493 / 503 staining on detecting the effect of interfering with E2F5 on the differentiation of ICP1 cell line; Figure 14 It is an analysis diagram of the effect of interfering with E2F5 on the expression of adipogenic differentiation-related genes. Among them, (A) is the analysis diagram of the expression (mean ± standard deviation) of adipogenic differentiation-related genes in ICP1 cells after interfering with E2F5 detected by qRT-PCR with NONO as the internal reference, (B) is the diagram of the expression of adipogenic differentiation-related genes in ICP1 cells after interfering with E2F5 detected by Western Blot with β-actin as the internal reference; (C) is the quantitative analysis diagram (mean ± standard deviation) of the protein level by western blot;

[0037] Figure 15 It is the diagram of the effect of overexpressing E2F5 on the activity of the promoter reporter gene of adipogenic differentiation-related genes;

[0038] Figure 16 It is the diagram of the effect of overexpressing E2F5 on the proliferation of ICP1 cells. Among them, (A) is the diagram of the effect of overexpressing E2F5 on the expression of cell proliferation marker genes in ICP1 cell line detected by qRT-PCR, (B) is the diagram of the effect of overexpressing E2F5 on the proliferation of ICP1 cell line detected by CCK-8, (C) is the diagram of the effect of overexpressing E2F5 on the proliferation of ICP1 cell line detected by EdU, and (D) is the statistical chart of the cell number of the effect of overexpressing E2F5 on the proliferation of ICP1 cell line detected by EdU.

[0039] Figure 17 It is the diagram of the effect of interfering with E2F5 on the proliferation of ICP1 cells. Among them, (A) is the diagram of the effect of interfering with E2F5 on the expression of cell proliferation marker genes in ICP1 cell line detected by qRT-PCR, (B) is the diagram of the effect of interfering with E2F5 on the proliferation of ICP1 cell line detected by CCK-8, (C) is the diagram of the effect of interfering with E2F5 on the proliferation of ICP1 cell line detected by EdU, and (D) is the statistical chart of the cell number of the effect of interfering with E2F5 on the proliferation of ICP1 cell line detected by EdU;

[0040] Figure 18 It is the diagram of the effect of overexpressing KLF7 and interfering with E2F5 on the differentiation of ICP1 cell line detected by Oil Red O staining;

[0041] Figure 19 It is the diagram of the effect of overexpressing KLF7 and interfering with E2F5 on the protein level of adipogenic differentiation-related genes detected by Western Blot;

[0042] Figure 20 It is a figure showing the effect of overexpressing KLF7 and interfering with E2F5 on the proliferation of ICP1 cell line detected by EdU. Specific implementation method:

[0043] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; unless otherwise specified, the materials, reagents, etc. used in the following examples can all be obtained from commercial channels.

[0044] Example 1:

[0045] The transcription factor E2F5 is used to regulate the differentiation of chicken preadipocytes. The regulation of the differentiation of chicken preadipocytes means inhibiting the differentiation of chicken preadipocytes by overexpressing the transcription factor E2F5. The transcription factor KLF7 plays a regulatory role by regulating the enhancer activity of the transcription factor E2F5. The sequence of the transcription factor E2F5 containing enhancer activity is as follows:

[0046] The binding sequence of the transcription factor KLF7 in the enhancer region of E2F5: ggaatgaaag;

[0047] The coding gene sequence of the transcription factor E2F5:

[0048] Preferably, the application method of the transcription factor E2F5 is as follows: construct the recombinant plasmid pCMV-HA-E2F5 and transfect chicken preadipocytes. The amino acid sequence of the recombinant plasmid pCMV-HA-E2F5 is as follows:

[0049] It is constructed by the recombination of the CDS sequence of the chicken E2F5 gene and the pCMV-HA eukaryotic expression vector. The construction method steps are as follows:

[0050] (1) Design of primers for amplifying the CDS region of the chicken E2F5 gene and PCR amplification,

[0051] Forward primer: tggccatgga ggcccgaatt ccggcgggca gcatgg;

[0052] Reverse primer: acacctacca agagtgctgt tcatac;

[0053] (2) Recovery of the E2F5 gene DNA fragment;

[0054] (3) Double digestion of the pCMV-HA eukaryotic expression vector;

[0055] (4) Ligation and transformation of the PCR amplification product of the chicken E2F5 gene CDS region with the linearized pCMV-HA eukaryotic expression vector;

[0056] (5) Small-scale extraction of plasmid DNA of the E2F5 gene;

[0057] (6) Identification of the pCMV-HA-E2F5 eukaryotic expression vector.

[0058] Example 2:

[0059] The following further illustrates the present invention with reference to the accompanying drawings.

[0060] 1.1 Unless otherwise specified, the test methods in the following examples are all conventional methods. Unless otherwise specified, the test materials used in the following examples are all obtained from conventional biochemical reagent companies.

[0061] 2.1 Cell culture and cell transfection

[0062] The ICP1 and DF-1 cell lines are cultured in DMEM medium (Gibco) supplemented with 10% fetal bovine serum (BI) and 1% penicillin-streptomycin solution (Solarbio) in a 37°C, 5% CO2 cell culture incubator.

[0063] When the cell confluence reaches 60-70%, transfection is carried out. The plasmid is extracted using a plasmid miniprep midiprep kit without endotoxin, and the Lipo8000 TM transfection reagent is used. Transfection is carried out according to the kit instructions. The plasmid is mixed with serum-free medium in the ratio specified in the instructions, added to the culture dish, shaken well, and then returned to the cell culture incubator for continued culture. The cell culture medium is changed once 6 h after transfection.

[0064] 2.2 Dual-luciferase activity detection

[0065] (1) Inoculate the ICP1 cell line or DF-1 cell line into a 24-well plate. When the cells reach 70-80% confluence, transfect the Renilla luciferase reporter gene vector pRL-TK and the recombinant plasmid, and set 3 replicates for each experimental group;

[0066] (2) 48 h after transfection, use a dual-luciferase reporter gene detection kit (TransGen Biotech, Beijing) to measure the luciferase activity according to the kit instructions.

[0067] 2.3 Gene cloning and vector construction

[0068] (1) The synthesized vectors are shown in Table 2.1.

[0069] Table 2.1 Experimental vectors

[0070]

[0071] (2) Design and synthesis of primers for chicken E2F5 gene promoter

[0072] Based on the chicken whole genome sequence ( https: / / genome.ucsc.edu / , UCSC) version Mar. 2018 (GRCg6a / galGal6), taking the first base A of the translation start codon ATG in the coding region of chicken E2F5 gene as +1, the upstream 5000 nt sequence was obtained. According to the prediction results of the potential promoter region of chicken E2F5 gene by Promoter 2.0, 4 pairs of primers for cloning the potential promoter of chicken E2F5 gene were designed using Primer Premier 5.0 and synthesized by Genewiz Biotechnology Co., Ltd. in Suzhou. The primer information and annealing temperature are shown in Table 2.2.

[0073] Table 2.2 Primers for cloning chicken E2F5 gene promoter

[0074]

[0075] (2) Cloning of chicken E2F5 gene promoter and construction of firefly luciferase reporter gene vector

[0076] Using the genomic DNA of AA white - feather broiler adipose tissue as a template, 4 chicken E2F5 gene promoter fragments were obtained by PCR amplification. Firefly luciferase reporter gene vectors pGL3 - E2F5(-971 / -183), pGL3 - E2F5(-1199 / -983), pGL3 - E2F5(-3464 / -1908) and pGL3 - E2F5(-4880 / -3624) were constructed using a homologous recombination kit (Vazyme) and named P1, P2, P3 and P4 respectively ( Figure 1 ).

[0077] High - fidelity DNA polymerase (Vazyme) was used for PCR amplification. The PCR reaction system was referred to the kit instruction manual. The reaction program was: 95℃ for 30 s, 40 cycles; 95℃ for 15 s, 62℃ for 15 s, 72℃ for 1 min, 72℃ for 5 min. 4 chicken E2F5 gene promoter fragments with corresponding homologous arms and restriction enzyme sites were obtained. The annealing temperature of each pair of primers is shown in Table 2.2. The PCR products were subjected to agarose gel electrophoresis at 150 V for 25 min. The target bands were cut out and recovered and transferred into a 10 mL centrifuge tube. The PCR products were purified and recovered using a gel extraction kit (TIANGEN) according to the kit instruction manual and the concentration was measured.

[0078] The pGL3-Basic empty vector was double-digested with the restriction endonucleases HindIII-HF (NEB) and KpnI-HF (NEB) to obtain the pGL3-Basic linearized vector. The double-digestion reaction system was as follows: 5 μL of CutSmart Buffer, 1000 ng of pCMV-Basic, 1 μL each of HindIII-HF and KpnI-HF, made up to 50 μL with ddH2O, reacted at 37 °C for 2 h, and then at 80 °C for 20 min. The PCR product was ligated to the linearized vector at 37 °C for 30 min using a homologous recombination kit (Vazyme, Nanjing) to construct a luciferase reporter gene vector for the potential promoter of the chicken E2F5 gene.

[0079] The next day, single colonies on the ampicillin-resistant plate were picked into 4 mL of ampicillin-resistant LB liquid medium in a clean bench and cultured at 37 °C in a shaker at 180 rpm for 8 - 10 h.

[0080] 300 μL of the bacterial solution was added with 100 μL of 80% glycerol for bacterial strain preservation. The plasmid was extracted from the bacterial solution using a plasmid miniprep kit (TIANGEN, Beijing) according to the kit instructions. The recombinant plasmids P1, P2, P3, and P4 were double-digested with the restriction endonucleases HindIII-HF (NEB) and KpnI-HF (NEB). The double-digested products were identified by agarose gel electrophoresis. The recombinant plasmid bacterial solution with correct identification was sent to Tianjin Genewiz Biotechnology Co., Ltd. for sequencing identification.

[0081] 2.4 Extraction of total RNA

[0082] The total RNA of the ICP1 cell line in a 6-well plate was extracted using the Trizol method. The specific operation was referred to the kit instructions.

[0083] 2.5 Reverse transcription of total RNA

[0084] Use II Q Select RT SuperMix for qPCR (+gDNA wiper) (Vazyme, Nanjing) for reverse transcription.

[0085] (1) The reaction system for removing genomic DNA was referred to the kit instructions. Reaction program: 42 °C for 2 min in a PCR instrument;

[0086] (2) Add 4 μL of 5×HiScript II Select qRT SuperMix II to the reaction solution in step (1) for reverse transcription reaction. After mixing, place it in a PCR instrument. The reaction program is as follows: 50 °C for 15 min; 85 °C for 5 s in the PCR instrument. Store the obtained cDNA in a -20 °C refrigerator and dilute it three-fold when used.

[0087] 2.6 qRT-PCR

[0088] (1) Design and synthesis of qRT-PCR primers

[0089] Design qRT-PCR primers for detecting chicken E2F5 expression using Primer Premier 5.0 and synthesize them by GenScript Biotech Corporation, Suzhou. The primer information is shown in Table 2.3.

[0090] Table 2.3 qRT-PCR primers

[0091]

[0092] (2) qRT-PCR reaction system

[0093] Using cDNA as a template, add the qRT-PCR primers shown in Table 2.3 and use ChamQ TM qPCR Master Mix (Vazyme, Nanjing) reagent. The reaction system refers to the kit instruction manual.

[0094] After mixing the above mixture evenly, place it in an Eppendorf ep realplex (Eppendorf, Foster City, Germany) for qRT-PCR. The reaction program refers to the kit instruction manual.

[0095] 2.7 Extraction of total cellular protein

[0096] Add 10 μL of PMSF to 1 mL of RIPA, mix well and place on ice; discard the cell culture medium in the well plate and wash it twice with PBS; add 100 μL of the prepared lysis solution to each well and lyse on ice for 15 - 25 min; scrape the cells with a spatula, transfer them to a centrifuge tube, centrifuge at 12,000 rpm at 4 °C for 5 min; aspirate the supernatant into a 1.5 mL centrifuge tube, mix it with 5×SDS protein loading buffer (Beyotime) at a ratio of 4:1, seal it with a sealing film, heat it in boiling water for 5 min, and store it at -20 °C.

[0097] 2.8 Western Blot

[0098] The eukaryotic expression vectors pCMV-Myc-KLF7 and pCMV-Myc were transfected into the ICP1 cell line. After continuous culture for 48 h, the cells were washed 3 times with PBS, and then 200 μL of RIPA lysis buffer (containing 1% PMSF) (Beyotime) was added to lyse the cells on ice for 15 min. The cells were scraped off and transferred to a 1.5 mL centrifuge tube, and centrifuged at 12,000 rpm for 5 min at 4 °C. The supernatant was the total protein. The protein sample was mixed with 5×SDS protein loading buffer (Beyotime) at a ratio of 4:1 and boiled for 5 - 10 min. The protein sample was stored in a -20 °C refrigerator. After electrophoresis at 90 V for about 30 min in a 12% separating gel and a 5% stacking gel, the voltage was changed to 150 V and run for 45 min - 1 h until the last Marker appeared, and then the electrophoresis was stopped. Transfer was carried out at 150 mA for 40 - 60 min. 5% non-fat milk blocking solution was added and blocked at room temperature for 2 h. Incubation was carried out with the primary antibody anti-HA (CST, USA, #3724, dilution ratio 1:1000) or anti-Myc (Beyotime, Shanghai, AM926, dilution ratio 1:1000) overnight at 4 °C. After washing 5 times with PBST, incubation with the anti-Rabbit secondary antibody was carried out for 1 h (LI-COR, USA, 680LT, dilution ratio 1:5000), and the membrane was scanned using an infrared imaging system. Then, the primary and secondary antibody removal solution was added and washed for 30 min, and blocked with 5% non-fat milk blocking solution at room temperature for 2 h. The internal reference antibody anti-β-actin (Beyotime, Shanghai, AF0003, dilution ratio 1:1000) was incubated overnight at 4 °C. After washing, the membrane was scanned using an infrared imaging system (LI-COR).

[0099] 2.9 Statistical analysis

[0100] The experiment was repeated 3 times, and the data were expressed as mean (Means) ± standard deviation (SD). Graphpad Prism 5.0 software was used for drawing, and the t-test was used to compare the differences between groups. P < 0.05 indicated significant differences, and P < 0.01 indicated extremely significant differences, which were considered to have statistical significance.

[0101] 2.10 Analysis of enhancer activity in the +22661 / +22900 region of the 3′ flanking region of the E2F5 gene

[0102] 2.10.1 Effect of the E2F5 (+22661 / +22900) region on the activity of the SV40 promoter

[0103] In the prior art, it was found by using ChIP-seq technology that there is a binding peak of KLF7 in the E2F5(+22661 / +22900) region, which is located in the 3′ flanking region of the gene and there is no promoter. Therefore, the +22661 / +22900 fragment of the 3′ flanking region of the E2F5 gene was ligated to the pGL3-promoter vector containing the SV40 promoter to construct the reporter gene vector pGL3-promoter-E2F5, which was transfected into the ICP1 and DF-1 cell lines. The results of the reporter gene showed that compared with the control group transfected with the empty vector, the dual luciferase activity of the experimental group transfected with pGL3-promoter-E2F5 was extremely significantly increased (P<0.01, Figure 2 ), indicating that the E2F5(+22661 / +22900) region can enhance the activity of the SV40 promoter, preliminarily suggesting that the E2F5(+22661 / +22900) region may have enhancer activity.

[0104] 2.10.2 Construction and activity analysis of the E2F5 gene promoter vector

[0105] Since most transcription factor regulatory elements are promoter-specific, the Promoter 2.0 was used to predict the potential promoter region of the chicken E2F5 gene ( Figure 1 A). According to the prediction results, 4 pairs of chicken E2F5 gene promoter cloning primers were designed (Table 2.4), and 4 chicken E2F5 gene promoter fragments were obtained by PCR amplification. The firefly luciferase reporter gene vectors pGL3-E2F5(-971 / -183), pGL3-E2F5(-1199 / -983), pGL3-E2F5(-3464 / -1908) and pGL3-E2F5(-4880 / -3624) were constructed and named P1, P2, P3 and P4 respectively ( Figure 1 B).

[0106] The recombinant plasmids P1, P2, P3 and P4 were identified by double digestion with the restriction endonucleases HindIII-HF and KpnI-HF. The results of agarose gel electrophoresis showed that the sizes of the target bands were 789bp, 218bp (the target band was not shown), 1557bp and 1257bp in turn ( Figure 3 ), and the sequencing results were correct. It was indicated that the firefly luciferase reporter gene vectors P1, P2, P3 and P4 were successfully constructed.

[0107] Four constructed firefly luciferase reporter gene vectors and the pGL3-Basic vector were co-transfected with the Renilla luciferase reporter gene vector pRL-TK into the ICP1 cell line, and the luciferase activity was detected 48 h after transfection. The results showed that in the ICP1 cell line, the promoter activity of P3 was extremely significantly lower than that of pGL3-Basic (P < 0.01, Figure 4 ), indicating that there is a negative regulatory element of promoter activity in the -3464 / -1908 region of the E2F5 gene; the promoter activity of P4 was extremely significantly higher than that of pGL3-Basic (P < 0.01, Figure 4 ), indicating that the -4880 / -3624 region of the E2F5 gene contains a promoter.

[0108] 2.10.3 The E2F5(+22661 / +22900) region can enhance the transcriptional activity of the E2F5 promoter

[0109] The E2F5(+22661 / +22900) fragment was ligated into the above-mentioned P4 vector with the promoter activity of the E2F5 gene to construct a P4-E2F5 enhancer reporter gene vector, which was co-transfected with the Renilla luciferase reporter gene vector pRL-TK into the ICP1 and DF-1 cell lines, and the luciferase activity was detected 48 h after transfection. The results showed that in the ICP1 and DF-1 cell lines, compared with the control group transfected with P4, the dual luciferase activity of the experimental group transfected with P4-E2F5 was extremely significantly increased (P < 0.01, Figure 5 ), indicating that after ligating the +22661 / +22900 fragment of the 3′ flanking region of the E2F5 gene, the luciferase activity of the E2F5 promoter can be significantly promoted, further indicating that the E2F5(+22661 / +22900) region may have enhancer activity.

[0110] 2.11 Regulation of the 3′ flanking region of the E2F5 gene by KLF7 and analysis of binding sites

[0111] The existing technology uses ChIP-seq technology and found a binding peak of KLF7 in the +22661 / +22900 region of the 3′ flanking region of the E2F5 gene, indicating that KLF7 may regulate E2F5 transcription. To further determine the regulatory effect and action site of KLF7 on the 3′ flanking region of the E2F5 gene, the two potential KLF7 binding sites in the +22661 / +22900 region fragment of the 3′ flanking region of the E2F5 gene were sequentially deleted, and the firefly luciferase reporter gene vectors pGL3-promoter-E2F5 (pGL3-E2F5), and the binding site deletion vectors pGL3-promoter-E2F5-DM1 and pGL3-promoter-E2F5-DM2 ( Figure 6 A) were synthesized respectively.

[0112] The three constructed reporter gene vectors were co-transfected with the eukaryotic expression vector pCMV-Myc-KLF7 and the Renilla luciferase reporter gene vector pRL-TK into the ICP1 and DF-1 cell lines, and the luciferase activity was detected 48 h after transfection. The results showed that compared with the control group co-transfected with pGL3-promoter-E2F5 and the pCMV-Myc empty vector, the relative luciferase activities of all experimental groups co-transfected with pGL3-promoter-E2F5 and pCMV-Myc-KLF7 were significantly increased (P<0.05, Figure 6 B, C); compared with the control group co-transfected with pGL3-promoter-E2F5-DM2 and pCMV-Myc, the relative luciferase activity of the experimental group co-transfected with pGL3-promoter-E2F5-DM2 and pCMV-Myc-KLF7 was significantly different (P<0.05, Figure 6 B, C), indicating that the deletion of DM2 could not completely eliminate the regulatory effect of KLF7 on the E2F5 gene, and KLF7 regulated the transcription of the E2F5 gene by binding to other sites; compared with the control group co-transfected with pGL3-promoter-E2F5-DM1 and pCMV-Myc, the relative luciferase activity of the experimental group co-transfected with pGL3-promoter-E2F5-DM1 and pCMV-Myc-KLF7 was not significantly different (P>0.05) and was significantly lower than that of the experimental group co-transfected with pGL3-promoter-E2F5 and pCMV-Myc-KLF7 (P<0.05, Figure 6 B, C), indicating that after the deletion of the binding site 1 "GGAATGAAAG" (+22727 / +22736 nt), KLF7 no longer up-regulated the activity of the 3'-flanking region of the E2F5 gene, suggesting that "GGAATGAAAG" (+22727 / +22736 nt) plays an important role in the regulation of E2F5 gene transcription by the transcription factor KLF7.

[0113] 2.12 Effect of overexpressing KLF7 on the endogenous expression of the E2F5 gene

[0114] To determine the effect of overexpressing KLF7 on the endogenous expression of the E2F5 gene in the ICP1 cell line, the eukaryotic expression vector pCMV-Myc-KLF7 and the pCMV-Myc empty vector were transfected into the ICP1 cell line respectively. After 48 h of transfection, the total protein of the ICP1 cell line was extracted, and the overexpression effect of KLF7 in the ICP1 cell line was detected by Western Blot using an anti-Myc tag antibody. The results showed that after 48 h of transfection, the eukaryotic expression vector pCMV-Myc-KLF7 expressed a specific protein band with a size of approximately 34 kD in the ICP1 cell line, while the pCMV-Myc empty vector did not ( Figure 7 A), indicating that KLF7 was successfully overexpressed in the ICP1 cell line.

[0115] The total RNA of the ICP1 cell line was extracted, and using TBP as the internal reference gene, the endogenous expression of E2F5 was detected by qRT-PCR. The results showed that compared with the control group transfected with the pCMV-Myc empty vector, overexpressing KLF7 could significantly up-regulate the relative expression level of E2F5 (P<0.05), indicating that overexpressing KLF7 in the ICP1 cell line could promote the transcription of E2F5 ( Figure 7 B). Combining with the reporter gene results indicated that E2F5 was a target gene of KLF7.

[0116] The results of this experiment confirmed that KLF7 promoted the transcription of the E2F5 gene, and "GGAATGAAAG" (+22727 / +22736 nt) played an important role in the transcription factor KLF7 regulating the transcription of the E2F5 gene; consistent with the reporter gene results, qRT-PCR confirmed that KLF7 promoted the endogenous expression of the E2F5 gene. Combining with the previous ChIP-seq results of the laboratory, it could be determined that E2F5 was a target gene of KLF7.

[0117] Example 3: Cloning of the coding sequence (CDS) of the chicken E2F5 gene and vector construction.

[0118] 3.1 Cloning of primers for cloning the coding region of the chicken E2F5 gene:

[0119] According to the chicken E2F5 gene sequence (NM_001030942.1) provided by the Genbank database on the NCBI website, primers for cloning the coding region of the chicken E2F5 gene were designed using Primer Premier 5.0 and synthesized by Suzhou Genewiz Biotechnology Co., Ltd. The primer information is shown in Table 3.1.

[0120] Table 3.1 Primers for cloning the coding region of the chicken E2F5 gene

[0121] Tab.3.1 Primers of chicken E2F5 gene coding sequence clone

[0122]

[0123] Using chicken preadipocyte cDNA as a template, PCR amplification was performed using high-fidelity DNA polymerase (Vazyme). The reaction system is shown in Table 2.3. The pCMV-HA empty vector was double-digested with restriction endonucleases EcoRI-HF (NEB) and XhoI (NEB) to obtain a linearized vector and its concentration was measured. The double-digestion reaction system is as follows: 5 μL CutSmart Buffer, 1000 ng pCMV-HA, 1 μL each of EcoRI and XhoI, and ddH2O was added to a final volume of 50 μL. The reaction was carried out at 37 °C for 2 h and then at 80 °C for 20 min. The PCR product was ligated to the linearized vector at 37 °C for 30 min using a homologous recombination kit (Vazyme, Nanjing). The ligation reaction system is shown in Table 2.4.

[0124] The double-digested product was subjected to agarose gel electrophoresis at 150 V for 25 min. The glycerol bacterial solution corresponding to the recombinant plasmid with correct double-digestion identification was sent to Tianjin Genewiz Biotechnology Co., Ltd. for sequencing identification.

[0125] 3.2 Design and synthesis of chicken E2F5 gene interference vector

[0126] The interference fragments sh-E2F5-1, sh-E2F5-2, sh-E2F5-3 and sh-NC of the E2F5 gene (NM_001030942.1) were designed using Invitrogen Block-iT RNAi Designer and synthesized by Suzhou Genewiz Biotechnology Co., Ltd. The fragment information is shown in Table 3.2. The lowercase letters in the table represent the cleavage sequences of restriction endonucleases EcoRI-HF and XhoI.

[0127] Table 3.2 sh-RNA sequences

[0128] Tab.3.2 sh-RNA sequences

[0129]

[0130] The upstream and downstream primers were annealed to form shRNA fragments and their concentrations were measured. The annealing reaction system was referred to the kit instructions. The reaction program was: 95 °C for 10 min, 37 °C for 1 h.

[0131] The pSilencer 1.0-U6 vector was linearized using the restriction endonucleases EcoRI-HF (NEB) and XhoI (NEB), and its concentration was measured. The shRNA fragments sh-E2F5-1, sh-E2F5-2, sh-E2F5-3, and sh-NC obtained after annealing were respectively ligated to the linearized pSilencer 1.0-U6 vector to construct E2F5 interference vectors, which were named sh-1, sh-2, sh-3, and sh-NC, respectively.

[0132] 3.3 In vitro induction of differentiation of ICP1 preadipocytes

[0133] When the ICP1 cells were cultured to about 40% confluence, they were induced to differentiate using a 160 μM oleic acid medium. The 160 μM oleic acid medium was changed every 24 h. After successful induction of cell differentiation, RNA was harvested and proteins were extracted at the corresponding time points.

[0134] 3.4 Oil Red O staining and extraction for colorimetry

[0135] After differentiation, the medium of ICP1 cells was discarded, and the cells were fixed with 4% paraformaldehyde at room temperature for 40 min. After drying in an oven at 65 °C for about 1 min, 1 mL of filtered Oil Red O working solution was added to each well, and the cells were stained for 50 min in the dark. The Oil Red O staining solution was aspirated, and the cells were washed three times with 1 mL of PBS; the PBS was discarded, 1 mL of 60% isopropanol was added and immediately aspirated, the surface floating color was washed off with PBS, 2 mL of PBS was added, and the lipid droplet staining was observed under a microscope. After taking pictures, the PBS was aspirated, 100 μL of isopropanol was added, and the cells were incubated at room temperature for 15 min, then diluted with three volumes of isopropanol, and the absorbance value at 510 nm was measured after extracting the dye.

[0136] 3.5 Extraction of total cellular RNA

[0137] See 2.4 for details

[0138] 3.6 Reverse transcription of total RNA

[0139] See 2.5 for details

[0140] 3.7 qRT-PCR

[0141] The steps are shown in detail in 2.6

[0142] Table 3.4 Primers for qRT-PCR

[0143] Tab.3.4 Primers for qRT-PCR

[0144]

[0145] 3.8 BODIPY 493 / 503 staining

[0146] For ICP1 preadipocytes, discard the culture medium, wash three times with PBS, dry the bottom of the dish slightly in the oven for 1 min, and fix on ice for 40 min. After washing three times with PBS, immediately aspirate after adding 60% isopropanol, add the working solution of BODIPY493 / 503 staining solution, and stain in the dark at room temperature for 15 min; wash three times with PBS, add Hoechst 33342 staining solution (diluted 1:1000), and stain in the dark at room temperature for 10 min. After washing three times with PBS buffer, take pictures using an inverted fluorescence microscope.

[0147] 3.9 Extraction of total cellular protein

[0148] See 2.2.7 for details.

[0149] 3.10 Western Blot

[0150] The steps are shown in detail in 2.2.8.

[0151] The primary antibodies used in the experiment mainly include: anti-KLF7 (Abmart, Shanghai, 6334-1, 1:200), anti-HA (CST, USA, #3724, dilution ratio 1:1000), anti-Myc (Beyotime, Shanghai, AM926, dilution ratio 1:1000), anti-C / EBPα (Abmart, Shanghai, 6223-1, 1:200), anti-FAS (Beyotime, Shanghai, AF6861, 1:1000), anti-PPARγ (Beyotime, Shanghai, catalog number, 1:1000), anti-FABP4 (Beyotime, Shanghai, AF6843, 1:1000). Using β-Actin as the internal reference, incubate with anti-β-actin (Beyotime, Shanghai, AF0003, dilution ratio 1:1000). The corresponding secondary antibodies are anti-Mouse (LI-COR, USA, 680RD, dilution ratio 1:5000) for β-actin and Myc-tagged antibodies, and the remaining antibodies' corresponding secondary antibodies are anti-Rabbit (LI-COR, USA, 680LT, dilution ratio 1:5000).

[0152] 3.11 Dual-luciferase activity assay

[0153] The method is the same as 2.2

[0154] 3.12 Statistical analysis

[0155] The method is the same as 2.9

[0156] 3.13 Expression pattern of E2F5 during the induction and differentiation of preadipocytes

[0157] The ICP1 cell line was seeded into a 6-well plate. When the density reached about 40% confluence, it was induced to differentiate using a 160 μM oleic acid medium. After 4 days of induction with oleic acid, the formation status of lipid droplets was observed under an inverted microscope. The results of Oil Red O staining and extraction colorimetry showed that small lipid droplets formed in the cells after 48 h of induction, and the absorbance value at 510 nm increased significantly, until the absorbance value at 96 h was 8 times that at 0 h ( Figure 8 A, B). Cellular RNA was extracted at 0, 12, 24, 48, 72, and 96 h after induced differentiation. Using NONO as an internal reference, the qRT-PCR results showed that the expression levels of the adipogenesis-related marker genes PPARγ, FABP4, FAS, and GPDH gradually increased with the increase of induction time ( Figure 8 C), indicating that the ICP1 cell line was successfully induced to differentiate. During the differentiation of preadipocytes, the expression level of the E2F5 gene decreased significantly with the increase of differentiation time (P<0.05, Figure 8 D), and this expression pattern suggested that E2F5 might play a negative regulatory role during the differentiation of chicken preadipocytes.

[0158] 3.14 Effects of overexpressing E2F5 on the differentiation of chicken preadipocytes

[0159] 3.14.1 Construction and verification of the E2F5 overexpression vector

[0160] To verify the role of E2F5 in the induced differentiation of preadipocytes, first, the pCMV-HA-E2F5 overexpression vector was constructed. The recombinant plasmid pCMV-HA-E2F5 was identified by double digestion with the restriction endonucleases EcoRI-HF and XhoI. The results of agarose gel electrophoresis showed that the size of the target band was 1095 bp ( Figure 9 A), and the sequencing results were correct, indicating that the pCMV-HA-E2F5 vector was successfully constructed. The Western Blot results showed that a specific protein band with a size of approximately 53.4 kD was expressed in the ICP1 cell line by the eukaryotic expression vector pCMV-HA-E2F5 after 48 h of transfection ( Figure 9 B), indicating that E2F5 was successfully overexpressed in the ICP1 cell line.

[0161] 3.14.2 Overexpressing E2F5 inhibits the formation of lipid droplets in chicken preadipocytes

[0162] To further confirm that E2F5 inhibits the differentiation of chicken preadipocytes, an Oil Red O binding extraction colorimetry experiment was performed on the lipid droplet content of ICP1 cells with overexpressed E2F5 in the ICP1 cell line. The results showed that compared with the control group, the lipid droplets in ICP1 cells with overexpressed E2F5 were extremely significantly reduced at the time points of 24, 48, and 72 h of induction (P<0.01,Figure 10 A, B), which was consistent with the results of Oil Red O staining. After 48 h of transfection, the results of BODIPY 493 / 503 staining showed that overexpression of E2F5 inhibited the number of lipid droplets during the induced differentiation of ICP1 preadipocytes ( Figure 10 C). The above results indicated that overexpression of E2F5 could inhibit lipid droplet formation in chicken preadipocytes.

[0163] 3.14.3 Overexpression of E2F5 inhibits the expression of adipogenic differentiation-related genes

[0164] The constructed overexpression vectors pCMV-HA-E2F5 and pCMV-HA empty vector were transfected into the ICP1 cell line. Using the anti-HA tag antibody, Western Blot was used to detect the overexpression effect of E2F5 in the ICP1 cell line. The results showed that after transfection for 24, 48, and 72 h, the eukaryotic expression vector pCMV-HA-E2F5 expressed a specific protein band with a size of approximately 53.4 kD in the ICP1 cell line, while the pCMV-HA empty vector did not ( Figure 11 B), indicating successful overexpression of E2F5 in the ICP1 cell line.

[0165] After induction of differentiation with 160 μM oleic acid medium, qRT-PCR was used to detect the effect of overexpression of E2F5 on the RNA level of adipogenic differentiation-related marker genes. The results showed that compared with the control group transfected with the empty vector, after overexpression of E2F5, the expressions of the adipogenesis-related genes C / EBPα, FAS, and FABP4 were significantly decreased at 48 h (P<0.05, Figure 11 A), among which the differences in C / EBPα and FABP4 were extremely significant at 72 h (P<0.01), and the expression of PPARγ was significantly decreased at 24 h (P<0.05).

[0166] Western Blot was used to detect the effect of overexpression of E2F5 on the protein level of adipogenic differentiation-related marker genes. The results showed that compared with the control group transfected with the empty vector, after overexpression of E2F5, the protein levels of the adipogenesis-related genes PPARγ, FAS, C / EBPα, and FABP4 showed a downward trend at each time point, and the expressions at 48 h were significantly lower than those of the control group transfected with pCMV-HA (P<0.05, Figure 11 B, C). Among them, C / EBPα was significant at 24 h (P<0.05), and the protein levels of PPARγ, C / EBPα, and FABP4 were significantly decreased at 72 h (P<0.05). Combining with the qRT-PCR results, it was suggested that overexpression of E2F5 could inhibit the protein expressions of the adipogenic differentiation-related genes PPARγ, FAS, C / EBPα, and FABP4.

[0167] 3.15 Effects of interfering with E2F5 on the differentiation of chicken preadipocytes

[0168] 3.15.1 Detection of the interference effect of chicken E2F5 gene shRNA

[0169] Three interference fragments were designed and synthesized according to the E2F5 gene sequence. After annealing, the obtained shRNA fragments sh-E2F5-1, sh-E2F5-2, and sh-E2F5-3 were respectively ligated to the linearized pSilencer 1.0-U6 vector to construct E2F5 interference vectors, which were named sh-1, sh-2, and sh-3 respectively. The sequencing results were consistent, indicating that the interference vectors were successfully constructed. Using the pSilencer 1.0-U6 empty vector and sh-NC vector as controls respectively, sh-1, sh-2, and sh-3 were transfected into the ICP1 cell line. After 48 h, total RNA of the ICP1 cell line was extracted. Using NONO as the internal reference gene, the interference effect of the E2F5 gene was detected by qRT-PCR.

[0170] The results showed that compared with the two control groups, the expression level of E2F5 in the experimental group transfected with sh-1 was extremely significantly decreased (P<0.01, Figure 12 ), so sh-1 was used for subsequent interference experiments.

[0171] 3.15.2 Interference of E2F5 promotes lipid droplet formation in chicken preadipocytes

[0172] In the ICP1 cell line, interference of E2F5 was performed, and an Oil Red O binding extraction colorimetric experiment was carried out on the lipid droplet content of ICP1 cells. The results showed that compared with the control group, the lipid droplets in ICP1 cells with E2F5 interference were extremely significantly increased at the 24, 48, and 72 h time points of induction (P<0.01, Figure 13 A, B), suggesting that interference of E2F5 can promote preadipocyte differentiation.

[0173] Consistent with the results of the Oil Red O experiment, BODIPY 493 / 503 staining after 48 h of transfection showed that interference of E2F5 can promote the number of lipid droplets during the induced differentiation of ICP1 preadipocytes ( Figure 13 C).

[0174] The above results indicate that interference of E2F5 can promote lipid droplet formation in chicken preadipocytes.

[0175] 3.15.3 Interference of E2F5 promotes the expression of genes related to fat generation

[0176] The qRT-PCR results showed that compared with the control group transfected with sh-NC, after interference of E2F5, the expression levels of the fat generation-related genes C / EBPα, PPARγ, and FABP4 increased at 48 and 72 h ( Figure 14A), in which both C / EBPα and FABP4 were significantly different at 48 and 72 h (P<0.05). The results suggest that interfering with E2F5 in the chicken ICP1 cell line can promote the expression of adipogenic differentiation-related genes such as PPARγ, C / EBPα, and FABP4, which are markers of adipogenesis.

[0177] Western Blot was used to detect the effect of interfering with E2F5 on the protein levels of adipogenic differentiation-related marker genes. The results showed that the protein levels of adipogenesis-related genes PPARγ, FAS, C / EBPα, and FABP4 in the experimental group with E2F5 interference were significantly higher at 48 h than those in the control group transfected with sh-NC (P<0.05, Figure 14 B, C). And there was an increasing trend in the protein levels of FAS, C / EBPα, and FABP4 at the remaining time points. Among them, FAS and C / EBPα were significant at 24 h (P<0.05), and FABP4 was significant at 72 h (P<0.05). Combining with the qRT-PCR results, it is suggested that interfering with E2F5 can promote the expression of adipogenic differentiation-related genes PPARγ, FAS, C / EBPα, and FABP4.

[0178] 3.16 Effect of overexpressing E2F5 on the promoter activity of adipogenic differentiation-related genes

[0179] To analyze the downstream genes of E2F5 in preadipocyte differentiation, E2F5 was overexpressed in ICP1 and DF-1 cell lines respectively, and the dual-luciferase reporter gene assay was used to analyze the promoter

[0180] activity of adipogenic differentiation-related genes. The results showed that overexpressing E2F5 significantly inhibited the promoter activities of PPARγ-1, PPARγ-2, and FAS (P<0.05, Figure 15 ), suggesting that E2F5 affects chicken preadipocyte differentiation through PPARγ and FAS.

[0181] 3.17 Conclusion

[0182] In the first few hours of adipocyte differentiation, the activities of E2F family members increase. This family is involved in cell cycle regulation during preadipocyte differentiation. For example, E2F1 and E2F3 are positive regulators of adipogenesis, while E2F4 negatively regulates adipogenesis, suggesting that E2F family members may play a role in the proliferation and differentiation of adipocytes. E2F5 was detected in human preadipocytes, but the role of E2F5 in adipocytes has not been reported. Therefore, experiments were conducted to analyze the function of E2F5 in the differentiation of chicken ICP1 preadipocytes.

[0183] As is well known, obesity is mainly due to the long-term imbalance between high energy intake and low energy consumption, resulting in excessive energy being stored in adipose tissue in the form of lipid droplets. Oil Red O is a fat-soluble dye that is highly soluble in fat and can specifically stain neutral fats such as triglycerides in the stained tissue or cells red. BODIPY 493 / 503 is a neutral lipid stain that emits a bright green fluorescence signal and can visually display lipid accumulation by exciting the fluorescence signal. It has been widely used in the staining experiments of adipocytes. In this study, it was first found that the expression level of E2F5 decreased significantly during differentiation. Oil Red O staining, extraction colorimetry, BODIPY 493 / 503 staining and other experiments were used to determine that E2F5 can inhibit the differentiation of chicken preadipocytes.

[0184] The expression of adipogenic differentiation-related genes after overexpression and interference of E2F5 was detected by qRT-PCR and Western Blot. It was preliminarily found that overexpression of E2F5 inhibited the expression of PPARγ, C / EBPα, FAS and FABP4, while interference with E2F5 gave the opposite results.

[0185] E2F1, E2F3 and E2F4 can bind to the promoter of PPARγ to regulate its activity and thus affect the differentiation of preadipocytes. In this experiment, it was found by dual-luciferase reporter gene assay that overexpression of E2F5 affected the differentiation of chicken preadipocytes by inhibiting the promoter activities of PPARγ and FAS, and this was consistent with the results of Western Blot; however, in the results of qRT-PCR, it was found that overexpression or interference of E2F5 mainly affected the expression levels of C / EBPα and FABP4.

[0186] The fat synthesis in chickens is different from that in mammals. For example, most of the triglycerides in mammals are synthesized in adipocytes, while the triglycerides in chickens are mainly synthesized by the liver; the fat formation mechanisms in mammals and birds are different, and the functions of the same transcription factors in fat formation in mammals and birds also vary. There are also significant differences between in vivo and in vitro experimental results. In the future, the CRISPR / Cas9 gene editing technology can be used to deeply study the function of E2F5 in the development of adipose tissue in chickens.

[0187] In summary, (1) the expression of E2F5 decreased significantly during the induced differentiation of chicken preadipocytes. (2) Overexpression of E2F5 inhibited the expression levels of fat formation-related genes and the accumulation of lipid droplets. (3) Interference with E2F5 promoted the expression levels of fat formation-related genes and the accumulation of lipid droplets. (4) Overexpression of E2F5 significantly inhibited the promoter activities of the adipogenic differentiation marker genes PPARγ-1, PPARγ-2 and FAS.

[0188] In conclusion, E2F5 inhibits the differentiation of chicken preadipocytes.

[0189] Example 4: Study on the Regulation of Chicken Preadipocyte Proliferation by E2F5

[0190] Members of the E2F family, such as E2F1, can promote the cell proliferation process in the G0 / S phase; the E2F4 / 5·p107 / p130 complex helps regulate the expression of the G0 / G1 cell cycle. As a transcriptional repressor of the E2F family members, the overexpression or amplification of E2F5 has been reported in solid tumors such as prostate cancer, liver cancer, esophageal cancer, ovarian cancer, and gastric cancer, but whether E2F5 plays a role in the process of adipocyte proliferation is still unclear. Therefore, this study explored the effect of E2F5 on the proliferation of ICP1 cell line by qRT-PCR, CCK-8, and EdU methods.

[0191] Table 4.1 qRT-PCR Primers

[0192] Tab.4.1 Primers for qRT-PCR

[0193]

[0194] 4.1 Detection of Cell Proliferation by CCK-8

[0195] The ICP1 cell line was inoculated into a 96-well plate, with 6 wells repeated in each experimental group. The transfection procedure was the same as in 2.2.4. The time when the cell line was inoculated for 6 h was recorded as 0 h. At 0, 24, 48, and 72 h, 10 μL of CCK-8 reagent (Dojindo) was added to each well in the dark, and after shaking well, it was incubated in a 37 °C, 5% CO2 cell culture incubator for 2 h. The absorbance value at 450 nm was measured using a microplate reader.

[0196] 4.2 Detection of Cell Proliferation by EdU

[0197] The ICP1 cell line was inoculated into a 6-well plate, with 3 wells repeated in each experimental group. The transfection procedure was the same as in 2.2.4. After 48 h of transfection, BeyoClick TMThe EdU-488 cell proliferation detection kit (Beyotime, Shanghai) was used to detect the proliferation of ICP1 cell line according to the kit instructions. After aspirating 1 mL of cell culture medium, 1 mL of 2×EdU-DMEM / F-12 cell culture medium preheated at 37°C was added, and after shaking well, it was incubated in a 37°C, 5% CO2 cell culture incubator for 2 h. 4% paraformaldehyde was added and fixed at room temperature for 15 min; the fixing solution was discarded, and after washing the cells 3 times with PBS, 0.3% PBST solution was added and permeabilized at room temperature for 10 min. 500 μL of Click reaction solution was added, and after shaking well, it was incubated in the dark at room temperature for 30 min; the cells were washed 3 times with PBS, and stained with 1×Hoechst33342 dye diluted with PBS in the dark at room temperature for 10 min. After washing the cells with PBS, they were observed and photographed under a fluorescence inverted microscope, and cell counting was performed using Image J.

[0198] 4.3 Experimental results

[0199] 4.3.1 Effect of overexpressing E2F5 on the proliferation of chicken adipocytes

[0200] To clarify the function of E2F5 in the proliferation of chicken ICP1 preadipocytes, the recombinant plasmid pCMV-HA-E2F5 and the pCMV-HA empty vector were transfected into ICP1 cells respectively, and total cellular RNA was harvested 48 h after transfection. Using NONO as an internal reference, the expression levels of proliferation marker genes were detected by qRT-PCR. The results showed that compared with the control group transfected with pCMV-HA, the proliferation marker gene ID2 was significantly increased after overexpressing E2F5 (P<0.05, Figure 16 A). The recombinant plasmid pCMV-HA-E2F5 and the pCMV-HA empty vector were transfected into ICP1 cells, and CCK-8 reagent was added at 0, 24, 48, and 72 h respectively, and the absorbance at 450 nm was measured using an enzyme-linked immunosorbent assay reader. The results showed that at 24, 48, and 72 h, the absorbance at 450 nm of the experimental group overexpressing E2F5 was extremely significantly higher than that of the control group (P<0.01, Figure 16 B). Detection using the BeyoClick TM EdU-488 cell proliferation reagent showed that the percentage of EdU-incorporated cells in the total number of cells in the E2F5 overexpression group was significantly higher than that in the control group (P<0.05, Figure 16 C, D). The above qRT-PCR, CCK-8, and EdU experimental results suggest that overexpressing E2F5 promotes the proliferation of ICP1 cell line.

[0201] 4.3.2 Effect of interfering with E2F5 on the proliferation of chicken adipocytes

[0202] To further clarify the function of E2F5 in the proliferation of chicken ICP1 preadipocytes, the interference vector sh-E2F5 and the sh-NC empty vector were transfected into ICP1 cells respectively, and the total cellular RNA was harvested 48 h after transfection. Using NONO as an internal reference, the expression levels of proliferation marker genes were detected by qRT-PCR. The results showed that compared with the transfection of the sh-NC empty vector, the expression levels of the cell proliferation marker genes Ki-67, PCNA, and CDK2 were significantly decreased after interfering with E2F5 (P<0.05, Figure 17 A), and the expression levels of Cyclin D1 and ID2 both showed a decreasing trend to varying degrees. The interference vector sh-E2F5 and the sh-NC empty vector were transfected into ICP1 cells, and the CCK-8 reagent was added at 0, 24, 48, and 72 h respectively. The absorbance at 450 nm was measured using an enzyme-linked immunosorbent assay reader. The results showed that at 24, 48, and 72 h, the absorbance at 450 nm of the experimental group with E2F5 interference was extremely significantly lower than that of the control group (P<0.01, Figure 17 B). The detection using the BeyoClick TM EdU-488 cell proliferation reagent showed that the percentage of EdU-incorporated cells in the E2F5 interference group was extremely significantly lower than that of the control group (P<0.01, Figure 17 C, D). The above qRT-PCR, CCK-8, and EdU experimental results suggest that interfering with E2F5 inhibits the proliferation of the ICP1 cell line.

[0203] 4.4 Conclusions

[0204] As is well known, the regulation of the cell cycle and the occurrence of apoptosis are classical molecular mechanisms of cell proliferation and growth. E2F family transcription factors play a central role in regulating cell proliferation by controlling the expression of genes required for cell cycle progression (especially DNA synthesis). Other transcription factors in the E2F family are involved in the differentiation of preadipocytes, yet there is no report on the function of E2F5 in the proliferation of preadipocytes.

[0205] To clarify the effect of E2F5 on the proliferation of preadipocytes cultured in vitro, E2F5 was overexpressed and interfered with in ICP1 cells. The qRT-PCR results showed that the expression of the proliferation marker gene ID2 was significantly upregulated after overexpressing E2F5 (P<0.05, Figure 16 ), and the expression of the proliferation marker genes Ki-67, PCNA, and CDK2 was significantly downregulated after interfering with E2F5 (P<0.05, Figure 17)。CCK-8 and EdU assay analysis found that overexpression of E2F5 promoted the proliferation of ICP1 cell line, while interference with E2F5 inhibited cell proliferation. It can be seen that E2F5 and KLF7 have the same function in chicken preadipocytes, both inhibiting preadipocyte differentiation and promoting cell proliferation. Since antibodies suitable for chicken Ki-67, PCNA, Cyclin D1, ID2 and CDK2 were not purchased, and antibodies of human or mouse Ki-67, PCNA, Cyclin D1, ID2 and CDK2 are not applicable to chicken research, the protein levels of the above proliferation marker genes in cells were not detected. Subsequently, it is necessary to further detect the effects of overexpression or interference of E2F5 on the protein levels of cell proliferation marker genes Ki-67, PCNA, Cyclin D1, ID2 and CDK2 in ICP1 cell line by Western Blot.

[0206] E2F transcription factors play a key role in regulating the cell cycle by controlling the transcription of genes encoding key components of the cell cycle and DNA replication mechanisms. As an oncogene, E2F1 can promote the cell proliferation process from G0 / S phase; chromatin immunoprecipitation experiments have shown that the E2F4 / 5·p107 / p130 complex helps regulate the expression of the early cell cycle including the G0 / G1 transition. The proliferation of preadipocytes is regulated by many genes. For example, miR-129-5p in 3T3-L1 preadipocytes inhibits cell proliferation by targeting and inhibiting the expression of G3BPs gene and blocking the transition of cells from S phase to G2 phase; bone morphogenetic protein 4 (BMP4) promotes the proliferation of chicken ICP1 preadipocytes by promoting the G1 / S phase transition. The results of this experiment confirmed that E2F5 promotes the proliferation of chicken preadipocytes. In future research, flow cytometry can also be used to detect the effect of E2F5 on the cell cycle of ICP1 cell line.

[0207] TP53 is also known as P53. Research has confirmed that E2F5 is involved in the carcinogenesis of breast cancer by inhibiting TP53 and has a proliferative effect on breast cancer; CircFAM13B upregulates the expression of E2F5 gene by recruiting miR-212 and activates the P53 pathway to promote the proliferation of liver cancer cells. Therefore, we speculate that during the proliferation of chicken preadipocytes, after KLF7 promotes the expression of E2F5 gene, it promotes the proliferation of chicken preadipocytes by activating the P53 signaling pathway. Subsequently, Western Blot can be used to detect the effects of overexpressing KLF7 and interfering with E2F5 on the protein levels of P53 target genes BAX, NOXA and PUMA for verification.

[0208] In summary, (1) after overexpressing E2F5, the expression level of the cell proliferation marker gene ID2 increased significantly, and the results of CCK-8 and EdU assays demonstrated that overexpressing E2F5 promoted the proliferation of chicken preadipocytes. (2) After interfering with E2F5, the expressions of the cell proliferation marker genes Ki-67, PCNA, and CDK2 decreased significantly, and the results of CCK-8 and EdU assays demonstrated that interfering with E2F5 inhibited the proliferation of chicken preadipocytes.

[0209] In conclusion, E2F5 promotes the proliferation of chicken preadipocytes.

[0210] Example 5: Study on the regulation of chicken preadipocyte proliferation and differentiation by KLF7 through E2F5

[0211] The differentiation and proliferation of the ICP1 cell line were detected using Oil Red O staining combined with extraction colorimetry, Western Blot assay, and EdU method to deeply study the mechanism of action of KLF7 during the development of chicken adipose tissue.

[0212] 5.1 KLF7 affects the differentiation of chicken preadipocytes through E2F5

[0213] 5.1.1 Oil Red O staining analysis shows that KLF7 inhibits the formation of lipid droplets in chicken preadipocytes through E2F5

[0214] KLF7 inhibits the differentiation of chicken preadipocytes

[11] , and this study confirmed that KLF7 promotes the expression of the E2F5 gene and E2F5 inhibits the differentiation of chicken preadipocytes. To determine whether KLF7 affects the differentiation of the ICP1 cell line through E2F5, the eukaryotic expression vector pCMV-Myc-KLF7 or the pCMV-Myc empty vector was co-transfected with the interference fragment sh-E2F5 or sh-NC into the ICP1 cell line. After 48 h of transfection, Oil Red O staining combined with extraction colorimetry and Western Blot assay were used to detect the formation of lipid droplets in the ICP1 cell line and the protein levels of adipogenic differentiation-related genes.

[0215] In the experimental group co-transfected with pCMV-Myc-KLF7 and sh-NC, the accumulation of lipid droplets was significantly less than that in the control group co-transfected with pCMV-Myc and sh-NC (P < 0.01, Figure 18 ), indicating that the differentiation degree of ICP1 cells was inhibited after overexpressing KLF7, suggesting that KLF7 inhibits the differentiation of the ICP1 cell line.

[0216] In the experimental group co-transfected with pCMV-Myc-KLF7 and sh-E2F5, the accumulation of lipid droplets was significantly higher than that in the control group co-transfected with pCMV-Myc-KLF7 and sh-NC (P < 0.01, Figure 18), and was similar to the lipid droplet accumulation in the experimental group co-transfected with pCMV-Myc and sh-NC (P>0.05, Figure 18 ), indicating that the inhibitory effect of KLF7 on the differentiation of ICP1 cells can be restored after interfering with E2F5, suggesting that KLF7 inhibits lipid droplet formation in the ICP1 cell line by promoting E2F5 gene transcription.

[0217] 5.1.2 Western Blot analysis of KLF7 inhibiting the protein expression of differentiation-related genes through E2F5

[0218] The Western Blot results showed that KLF7 was successfully overexpressed ( Figure 19 ). The protein levels of adipogenic differentiation-related genes C / EBPα, FAS, and FABP4 in the experimental group co-transfected with pCMV-Myc-KLF7 and sh-NC were significantly lower than those in the control group co-transfected with pCMV-Myc and sh-NC (P<0.05, Figure 19 ), indicating that overexpressing KLF7 can inhibit the protein expression levels of adipogenic differentiation-related genes, suggesting that KLF7 inhibits the differentiation of the ICP1 cell line.

[0219] The protein levels of adipogenic differentiation-related genes PPARγ, FAS, and FABP4 in the experimental group co-transfected with pCMV-Myc-KLF7 and sh-E2F5 were significantly higher than those in the control group co-transfected with pCMV-Myc-KLF7 and sh-NC (P<0.05, Figure 19 ), and there was no significant difference in protein expression levels compared with the experimental group co-transfected with pCMV-Myc and sh-NC (P>0.05, Figure 19 ), indicating that the inhibitory effect of KLF7 on the protein expression of adipogenic differentiation-related genes can be restored after interfering with E2F5, suggesting that KLF7 inhibits the reduction of lipid accumulation by promoting E2F5 gene transcription to inhibit the protein expression of adipogenic differentiation-related genes, thereby inhibiting the differentiation of chicken preadipocytes.

[0220] 5.2 KLF7 affects the proliferation of chicken preadipocytes through E2F5

[0221] KLF7 promotes the proliferation of chicken preadipocytes. This study confirmed that KLF7 promotes E2F5 gene expression and E2F5 promotes the proliferation of chicken preadipocytes. To determine whether KLF7 promotes the proliferation of the ICP1 cell line through E2F5, the eukaryotic expression vector pCMV-Myc-KLF7 or the pCMV-Myc empty vector was co-transfected with the interference fragment sh-E2F5 or sh-NC into the ICP1 cell line. After 48 h of transfection, the BeyoClick TM EdU-488 cell proliferation detection kit was used to detect the proliferation of the ICP1 cell line.

[0222] The percentage of EdU-incorporated cells in the experimental group co-transfected with pCMV-Myc-KLF7 and sh-NC was extremely significantly higher than that in the control group co-transfected with pCMV-Myc and sh-NC (P < 0.01, Figure 20 ), indicating that overexpression of KLF7 increased the amount of DNA synthesis in ICP1 cells, suggesting that KLF7 promoted the proliferation of the ICP1 cell line.

[0223] The percentage of EdU-incorporated cells in the experimental group co-transfected with pCMV-Myc-KLF7 and sh-E2F5 was extremely significantly lower than that in the control group co-transfected with pCMV-Myc-KLF7 and sh-NC (P < 0.01, Figure 20 ), and there was no significant difference in the percentage of EdU-incorporated cells in the experimental group co-transfected with pCMV-Myc-KLF7 and sh-E2F5 compared with that in the experimental group co-transfected with pCMV-Myc and sh-NC (P > 0.05, Figure 20 ), indicating that interfering with E2F5 could restore the promoting effect of KLF7 on the proliferation of the ICP1 cell line, suggesting that KLF7 promoted the proliferation of the ICP1 cell line by promoting E2F5 gene transcription.

[0224] 5.3 Conclusion

[0225] KLF7 inhibited the differentiation of chicken preadipocytes and promoted their proliferation. In adipose tissue, KLF7 inhibited the differentiation of chicken preadipocyte cell lines by promoting GATA3 and inhibiting HIF1α gene transcription, and KLF7 promoted the proliferation of ICP1 cells by promoting CDKN3 gene transcription.

[0226] In this study, the Oil Red O experiment found that interfering with E2F5 could restore the inhibitory effect of KLF7 on the differentiation of ICP1 cells, and the Western Blot experiment results found that interfering with E2F5 could restore the inhibitory effect of KLF7 on the protein expression of adipogenic differentiation-related genes, indicating that KLF7 inhibited the protein expression of adipogenic differentiation-related genes and reduced lipid accumulation by promoting E2F5 gene transcription, thereby inhibiting the differentiation of chicken preadipocytes. In Chapter 3, it was found that overexpression of E2F5 inhibited the promoter activities of PPARγ-1, PPARγ-2 and FAS and the expression of PPARγ and FAS. It was thus speculated that KLF7 affected the expression of PPARγ and FAS by regulating E2F5, and further inhibited the differentiation of chicken preadipocytes.

[0227] The EdU experiment found that the promotion of the proliferation of the ICP1 cell line by KLF7 could be restored after interfering with E2F5, indicating that KLF7 promotes the proliferation of the ICP1 cell line by promoting the transcription of the E2F5 gene. KLF7 affects the differentiation and proliferation of the ICP1 cell line by promoting the transcription of the E2F5 gene. In the future, the CRISPR / Cas9 gene editing technology can be used to deeply study the mechanism of action of KLF7 regulating E2F5 in the development of chicken adipose tissue.

[0228] Approximately 60% of the genes in chickens are highly homologous to those in humans. Chickens are potential models for studying human obesity and obesity-related diseases. The research results contribute to improving the molecular regulation network of adipose tissue development, provide important references for deeply studying the functions of KLF7 and E2F5 in adipose tissue development, offer new ideas for the research of other members of the KLFs family, and also provide references for further understanding the molecular mechanisms of human adipogenesis and obesity occurrence, which is of great significance for preventing obesity.

[0229] In summary, (1) The results of Oil Red O binding extraction colorimetric experiment and Western Blot experiment confirmed that interfering with E2F5 could restore the inhibitory effect of KLF7 on the differentiation of ICP1 cells, indicating that KLF7 inhibits the differentiation of chicken preadipocytes through E2F5. (2) The results of the EdU experiment confirmed that interfering with E2F5 could restore the promoting effect of KLF7 on the proliferation of ICP1 cells, indicating that KLF7 promotes the proliferation of chicken preadipocytes through E2F5.

[0230] In short, KLF7 affects the differentiation and proliferation of chicken preadipocytes through E2F5.

Claims

1. Use of a transcription factor E2F5, characterized in that, It is used to regulate the differentiation of chicken preadipocytes.

2. The use according to claim 1, characterized in that, The regulation of the differentiation of chicken preadipocytes refers to inhibiting the differentiation of chicken preadipocytes by overexpressing the transcription factor E2F5.

3. The use according to claim 1 or 2, characterized in that, The transcription factor KLF7 plays a regulatory role by regulating the enhancer activity of the transcription factor E2F5. The sequence of the transcription factor E2F5 containing enhancer activity is shown in SEQ ID No.1, and the sequence where the transcription factor KLF7 binds to the enhancer region of E2F5 is shown in SEQ ID No.

2.

4. A transcription factor E2F5 for regulating chicken fat formation, characterized in that, The coding gene sequence of the transcription factor E2F5 is shown in SEQ ID No.

3.

5. A method for applying the use according to any one of claims 1-4, characterized in that, Construct the recombinant plasmid pCMV-HA-E2F5 and transfect chicken preadipocytes. The amino acid sequence of the recombinant plasmid pCMV-HA-E2F5 is shown in SEQ ID No.

4.

6. A plasmid for overexpressing chicken E2F5 gene, characterized in that it is constructed by recombining the CDS sequence of chicken E2F5 gene with the pCMV-HA eukaryotic expression vector; the construction method steps are as follows: (1) Design of primers for amplifying the CDS region of chicken E2F5 gene and PCR amplification, Forward primer: as shown in SEQ ID NO.5; Reverse primer: as shown in SEQ ID NO.6; (2) Recovery of the E2F5 gene DNA fragment; (3) Double digestion of the pCMV-HA eukaryotic expression vector; (4) Ligation and transformation of the PCR amplification product of the CDS region of chicken E2F5 gene with the linearized pCMV-HA eukaryotic expression vector; (5) Small-scale extraction of the plasmid DNA of E2F5 gene; (6) Identification of the pCMV-HA-E2F5 eukaryotic expression vector.

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