WO2022121509A1 - 一种调控脂肪细胞形成的转录因子c/ebpz及其应用 - Google Patents

一种调控脂肪细胞形成的转录因子c/ebpz及其应用 Download PDF

Info

Publication number
WO2022121509A1
WO2022121509A1 PCT/CN2021/124592 CN2021124592W WO2022121509A1 WO 2022121509 A1 WO2022121509 A1 WO 2022121509A1 CN 2021124592 W CN2021124592 W CN 2021124592W WO 2022121509 A1 WO2022121509 A1 WO 2022121509A1
Authority
WO
WIPO (PCT)
Prior art keywords
ebpz
chicken
transcription factor
adipocytes
gene
Prior art date
Application number
PCT/CN2021/124592
Other languages
English (en)
French (fr)
Inventor
张志威
陈月婵
曹冬冬
高玲羽
林涛
Original Assignee
石河子大学
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 石河子大学 filed Critical 石河子大学
Publication of WO2022121509A1 publication Critical patent/WO2022121509A1/zh
Priority to ZA2023/03608A priority Critical patent/ZA202303608B/en

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/124—Animal traits, i.e. production traits, including athletic performance or the like
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the invention relates to the fields of animal molecular genetics and developmental biology, in particular to a transcription factor C/EBPZ that regulates the formation of adipocytes and its application.
  • Abdominal adipose tissue also known as visceral adipose tissue, is located in the abdominal cavity and surrounds the gastrointestinal organs and other organs; it is mainly composed of white adipocytes, which are important energy storage and endocrine organs in animals. .
  • Abdominal adipose tissue deposition mainly occurs during the growth and development stage of broilers.
  • Adipocyte formation is the direct cause of adipose tissue deposition.
  • adipocyte precursors mainly exist in the adipose stromal-vascular fraction (SVF).
  • SVF adipose stromal-vascular fraction
  • VLDL plasma very low density lipoprotein
  • two-way selection of abdominal fat content to establish high and low abdominal fat content broiler groups, as well as the commercial generation of "fast food" widely used in broiler production
  • Large "broiler chickens are suitable animal models for the study of abdominal fat deposition in chickens.
  • extracellular signaling molecules such as neuropeptide Y (NPY) and BMP4, transcriptional molecules such as PPAR ⁇ , C/EBP ⁇ , SREBP1, KLF2, KLF3, KLF5 and KLF7 Factors, as well as miRNAs such as gga-miR-21, have important regulatory roles in chicken adipocyte formation.
  • extracellular signaling molecules such as neuropeptide Y (NPY) and BMP4, transcriptional molecules such as PPAR ⁇ , C/EBP ⁇ , SREBP1, KLF2, KLF3, KLF5 and KLF7 Factors, as well as miRNAs such as gga-miR-21, have important regulatory roles in chicken adipocyte formation.
  • C/EBPs CCAAT/enhancer binding proteins
  • bZIP basic leucine zipper
  • the name was coined by Steven L McNight, named after the first identified C/EBP protein with several promoter CCAAT boxes (CCAAT boxes) and some viral enhancers the ability to bind to the core homology region.
  • CCAAT boxes CCAAT boxes
  • a total of six C/EBP family members have been found in mammals: C/EBP ⁇ , C/EBP ⁇ , C/EBP ⁇ , C/EBP ⁇ , C/EBP ⁇ and DDIT3.
  • the CCAAT box was one of the first DNA elements discovered to regulate eukaryotic gene expression, and early studies reported that C/EBPs could bind to the CCAAT box. However, recent research reports show that C/EBPs do not recognize the CCAAT box, and the site they recognize is TT(G/A)CGCAA, not GGCCAATCT.
  • C/EBPZ CCAAT/enhancer binding protein zeta
  • C/EBPZ is a special gene that does not belong to the C/EBP family in a strict sense because it does not have the typical basic leucine zipper structure.
  • C/EBPZ also known as CBF, CBF-2, NOC1, and HSP-CBF, was first discovered when studying the transcriptional regulation of human HSP70.
  • the purpose of the present invention is to provide a transcription factor C/EBPZ that regulates the formation of adipocytes and its application, so as to solve the above-mentioned problems in the prior art.
  • the present invention provides the following scheme:
  • the present invention provides a transcription factor C/EBPZ that regulates the formation of chicken adipocytes, and the coding gene sequence of the transcription factor C/EBPZ is shown in SEQ ID NO.1.
  • the present invention also provides a use of the transcription factor C/EBPZ in preparing a preparation or composition for regulating chicken adipocyte formation.
  • the regulation of chicken adipocyte formation refers to the regulation of the differentiation and proliferation of chicken adipocytes.
  • the regulation of chicken adipocyte differentiation means that overexpression of transcription factor C/EBPZ inhibits chicken adipocyte differentiation.
  • the regulation of chicken adipocyte proliferation means that overexpression of transcription factor C/EBPZ promotes chicken adipocyte proliferation.
  • the gene regulating chicken adipocyte formation is selected from PPAR ⁇ , FASN, C/EBPa, LPL, FABP4, GATA2 or a combination thereof.
  • promoter activity and mRNA expression of the gene were regulated by overexpression of transcription factor C/EBPZ in chicken adipocytes.
  • the present invention also provides a use of the transcription factor C/EBPZ in screening broilers.
  • described screening is specifically: detect the mRNA expression level of C/EBPZ gene in the abdominal adipose tissue of broiler chicken, detect the nucleotide sequence of the primer pair used for C/EBPZ gene expression such as SEQ ID NO.3 and SEQ ID NO.4 shown.
  • the present invention provides a recombinant plasmid pCMV-HA-C/EBPZ, the recombinant plasmid pCMV-HA-C/EBPZ includes the transcription factor C/EBPZ, and the nucleotides of the recombinant plasmid pCMV-HA-C/EBPZ The sequence is shown in SEQ ID NO.2.
  • the invention discloses the application of a transcription factor C/EBPZ in regulating the formation of chicken adipocytes.
  • the real-time PCR technology is used to detect the mRNA expression level of C/EBPZ gene in chicken abdominal adipose tissue, which verifies that the gene is in There were significant differences in the expression levels in abdominal adipose tissue of high- and low-fat broilers.
  • an overexpression vector of the transcription factor was constructed to verify that C/EBPZ transcription factor regulates chicken preadipocyte differentiation and chicken preadipocyte proliferation. Mechanism of action in adipose tissue formation.
  • This transcription factor affects the proliferation and differentiation of chicken preadipose by regulating the expression of PPAR ⁇ , FASN, C/EBP ⁇ , LPL, FABP4, GATA2 and KLF2, etc. or their combinations.
  • the invention provides a new application of C/EBPZ transcription factor in regulating the formation of chicken adipocytes, and has practical value in the fields of chicken genetics breeding and animal nutrition.
  • Fig. 1 is the plasmid map of C/EBPZ overexpression vector (pCMV-HA-C/EBPZ) and empty vector (pCMV-HA);
  • Figure 2 shows the expression pattern of C/EBPZ gene in various tissues of broilers; among them: 1: abdominal fat, 2: brain, 3: duodenum, 4: gizzard, 5: heart, 6: ileum, 7: jejunum, 8: stomach, 9: leg muscle, 10: liver, 12: breast muscle, 13: glandular stomach, 14: spleen, 15: testis;
  • Fig. 4 is the plasmid expression protein verification of C/EBPZ overexpression vector
  • Figure 5 is a graph showing the effect of overexpression of C/EBPZ on the proliferation of chicken preadipocytes
  • Figure 6 is a graph showing that overexpression of C/EBPZ inhibits the differentiation of chicken preadipocytes
  • Figure 7 is a graph showing the overexpression of C/EBPZ to regulate the promoter activity of PPAR ⁇ , FASN, C/EBP ⁇ , LPL, FABP4 and GATA2;
  • Figure 8 shows the enrichment analysis of transcriptome gene sets in chicken adipocytes after C/EBPZ overexpression for 48 hours;
  • A is the number of differential genes of overexpressed C/EBPZ compared with the control group shown by RNA-seq analysis;
  • B is the comparison of RNA-seq result data for gene set enrichment analysis map;
  • Figure 9 shows the real-time PCR results of the effect of overexpression of C/EBPZ on the expression of endogenous adipocyte formation marker genes in chicken preadipocytes, which was done to verify the results of RNA-seq.
  • abdominal fat pads were collected after slaughter.
  • 15 other tissues including liver, duodenum, jejunum, ileum, thorax, leg muscles, perigastric fat, heart, spleen, kidney, pancreas, glandular stomach, brain, and testis were also collected . All collected tissues were immediately frozen in liquid nitrogen and stored in a -80°C freezer for later use.
  • reaction system I total RNA 1.0 ⁇ g, oligod T primer (2.5 ⁇ M) 0.5 ⁇ L, and RNase free ddH 2 O was added to make up 5 ⁇ L .
  • the above reaction system I was treated at 70°C for 5 min, and then placed on ice for 5 min to destroy the secondary structure of RNA, and then the reaction system II was configured as follows:
  • the reverse transcription conditions are as follows:
  • CEBPZ CF GTCGACCATGGCGGCGCTCGGGGAGT
  • CEBPZ CR AGATCTTCATCTTTTTGATTTCTTGCCTC
  • the reaction system is as follows:
  • PCR amplification conditions were: pre-denaturation at 94°C for 7 min; denaturation at 94°C for 30 s, annealing at 62°C for 30 s, extension at 72°C for 2 min, a total of 35 cycles, final extension at 72°C for 7 min, and termination of the reaction at 4°C.
  • the PCR product was subjected to agarose gel electrophoresis to recover the target band.
  • the target gene was ligated to the pMD-18T vector (Takara), the ligated product was transferred into Escherichia coli DH5 ⁇ , AMP resistance was screened and cultured, a single clone was picked, inoculated into LB liquid medium, the plasmid was extracted, and the plasmid was sequenced , the C/EBPZ nucleotide sequence inserted into pMD-18T was measured as shown in SEQ ID NO.1, and the obtained positive recombinant plasmid was marked as pMD-18T-C/EBPZ.
  • the two endonucleases SalI and BglII were used for double digestion.
  • the system is as follows:
  • the digestion conditions were: 37°C for 1 h.
  • the digested product was identified by 1% agarose gel electrophoresis and purified and recovered by AXYGEN gel recovery and purification kit.
  • T4 DNA ligase was used to connect the target gene fragment and backbone vector fragment together to construct pCMV-HA-C/EBPZ vector , the ligated product was transferred into E. coli DH5 ⁇ , AMP resistance was screened for culture, the single clone was picked out, the single clone was inoculated in LB liquid medium, the plasmid was extracted, and the positive recombinant plasmid was obtained by double digestion and sequencing with SalI and BglII.
  • pCMV-HA-C/EBPZ the sequence of the recombinant plasmid is shown in SEQ ID NO.2.
  • the plasmid maps of the C/EBPZ overexpression vector (pCMV-HA-C/EBPZ) vector and the empty vector (pCMV-HA) are shown in Figure 1.
  • Real-time PCR was completed using SYBR Premix Ex Taq kit (Takara company) and ABI Prism 7500 sequence detection system (Applied Biosystems company), the reaction used 20 ⁇ L system, the reaction system was configured on ice, the system includes: cDNA 2 ⁇ L, 2 ⁇ SYBR Premix Ex Taq 10 ⁇ L, PCR Forward (Reverse) Primer (10 ⁇ mol/L) 0.4 ⁇ L each, 50 ⁇ Rox Reference DyeII 0.4 ⁇ L, double distilled water 6.8 ⁇ L; the reaction conditions were pre-denaturation at 95°C for 5s, and then 40 cycles were carried out. 1 cycle included 95°C for 5s and 60°C for 34s; after 40 cycles were completed, the dissolution curve (dissociation curve) was detected.
  • the primers used to analyze the expression of C/EBPZ are shown in SEQ ID NO.3 and SEQ ID NO.4.
  • the results showed that C/EBPZ was widely expressed in a variety of chicken tissues (Figure 2), and the relative expression level of C/EBPZ (C/EBPZ/ ⁇ -ACTIN) in the abdominal adipose tissue of low-fat broilers was significantly higher than that of high-fat chickens Line broiler (Fig. 3A, P ⁇ 0.01), and the relative expression level of C/EBPZ mRNA in abdominal adipose tissue of low-fat broiler chickens at the 7th and 9th week of age was significantly higher than that of high-fat line broiler (Fig. 3B, P ⁇ 0.05) . It is suggested that the relative expression level of C/EBPZ mRNA (C/EBPZ/ ⁇ -ACTIN) can be used as a molecular marker of abdominal fat content.
  • Abdominal adipose tissue (3-5 g) was taken from 12-day-old AA broilers, washed twice with PBS, digested with 2 mg/mL type I collagenase (Sigma) at 37°C for 1 hour, up and down every 10 minutes Mix by inversion once.
  • vascular stromal (SVF) cells derived from chicken abdominal adipose tissue are used as chicken preadipocytes.
  • the isolated chicken preadipocytes were suspended in complete medium (DMEM/F12+10%FBS+1%K), inoculated into a cell culture flask at a density of 1 ⁇ 10 5 cells/cm 2 , and kept at 37° C., Incubate under 5% CO 2 conditions.
  • complete medium DMEM/F12+10%FBS+1%K
  • the chicken preadipocytes in good growth condition were seeded into 6-well cell culture plates at a seeding density of 1 ⁇ 10 5 cells/well. After 24 hours of passage, about 70-90% of the cells were confluent. According to the instructions of Fugene HD (Promega)
  • the empty expression vector plasmid pCMV-HA and C/EBPZ overexpression plasmid pCMV-HA-C/EBPZ were respectively transfected into chicken preadipocytes cultured in 6-well plates, and 2.0 ⁇ g of plasmid was transfected in each well, and the cells were recovered 48 hours after transfection.
  • the chicken preadipocytes in good growth condition were seeded into 6-well cell culture plates at a seeding density of 1 ⁇ 10 5 cells/well. After 24 hours of passage, about 70-90% of the cells were confluent. Plasmids pCMV-HA and pCMV-HA-C/EBPZ were transfected into chicken preadipocyte cells in different wells. After 24 hours of growth, the adherent cells were digested with trypsin and inoculated into 96 cells at a concentration of 5000 cells per well. In the well cell culture plate, 24h, 48h, 72h, 96h and 120h after inoculation, using The number of cell proliferation was detected by the luminescence cell viability detection kit (Promega). The results are shown in Figure 5.
  • the chicken preadipocytes in good growth condition were seeded into 6-well cell culture plates at a density of 1 ⁇ 10 5 cells/well. After 24 hours of passage, about 70-90% of the cells were confluent. Fugene HD transfection reagent (Promega) was used.
  • the plasmids pCMV-HA and pCMV-HA-C/EBPZ were transfected into chicken preadipocyte cells in different wells according to the instructions. About 48h after transfection, when the cells in the cell culture plate were completely confluent (the cells were 100% confluent) ), preadipocytes were induced to differentiate into adipocytes with 160 nM sodium oleate (Sigma).
  • the chicken preadipocytes in good growth condition were inoculated into a 12-well cell culture plate at a seeding density of 5 ⁇ 10 4 cells/well. After 24 hours, each group of plasmids were transfected into chicken preadipocytes according to Fugene HD (Promega) instructions. , the grouping looks like this:
  • Well-grown chicken preadipocytes were seeded into 6-well plates at a density of 1 ⁇ 10 5 cells/cm 2 . After 12 hours of inoculation, the cells were about 60-80% confluent, and the cells were transfected with pCMV-HA-C/EBPZ or empty vector (pCMV-HA) plasmid according to Fugene HD (Promega) transfection reagent according to the instructions, 48h after transfection, cells were collected, and Tianjin Nuohezhiyuan Bio-Information Technology Co., Ltd. was entrusted to conduct transcriptome RNA-seq analysis.
  • RNA-seq results shown in Figure 8A compared with the control group, after overexpression of C/EBPZ, 448 genes were up-regulated and 354 genes were down-regulated; as shown in Figure 8B, gene set enrichment analysis (gene set enrichment analysis) , GSEA) showed that the expression of adipose tissue developmental gene (ADIPOSE_TISSUE_DEVELOPMENT, GO: 0060612)) was significantly up-regulated (P ⁇ 0.01) after overexpression of C/EBPZ in chicken preadipocytes, indicating that overexpression of C/EBPZ is involved in the regulation of adipocyte formation.
  • GSEA gene set enrichment analysis
  • Well-grown chicken preadipocytes were seeded into 6-well plates at a density of 1 ⁇ 10 5 cells/cm 2 . 12 hours after inoculation, the cells grew to about 60-80% confluence, and the cells were transfected with pCMV-HA-C/EBPZ or empty vector (pCMV-HA) plasmid according to Fugene HD (Promega) transfection reagent according to the instructions. Cells were harvested 48 h after transfection.
  • Adherent chicken preadipocytes were collected with TRIzol reagent and total RNA was extracted therefrom. The concentration of extracted RNA was determined with Nano drop 2000 (Thermo scientific), and 1 ⁇ g RNA was reversed using Promega-Improm II (Promega) reverse transcription reagent kit. cDNA was transcribed under reverse transcription conditions: 25.0°C for 5 min, 42.0°C for 60 min, 70.0°C for 15 min, and incubation at 5.0°C.
  • the system includes: 5 ⁇ L of 2 ⁇ QuantiNova SYBR Green RT-PCR Master Mix, 0.2 ⁇ L of PCR Forward (Reverse) Primer (10 ⁇ mol/L), 1 ⁇ L of cDNA product, 3.6 ⁇ L of double distilled water .
  • Real time PCR analysis was performed using QIAGEN qRT-PCR instrument system (QIAGEN, Hilden, Germany), the program was set as: 95.0 °C for 5 min, then 40 cycles: 95.0 °C for 10 s, 56.0 °C for 30 s, 72.0 °C for 40 s plus The dissociation curve was analyzed using Rotor-Gene Q Series Software 2.3.1 software.
  • the primers used are shown in Table 1 below:

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Analytical Chemistry (AREA)
  • Zoology (AREA)
  • Biochemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • Molecular Biology (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Toxicology (AREA)
  • Physics & Mathematics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biotechnology (AREA)
  • Immunology (AREA)
  • Microbiology (AREA)
  • Medicinal Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

提供了一种调控脂肪细胞形成的转录因子C/EBPZ及其应用，具体为转录因子C/EBPZ调控鸡脂肪细胞形成的用途。利用RT-PCR技术揭示了鸡C/EBPZ在多种组织中的表达模式，和鸡脂肪组织生长发育过程中的C/EBPZ的表达规律。构建了该转录因子的过表达载体，验证了C/EBPZ转录因子可调控鸡前脂肪细胞的分化和增殖。

Description

一种调控脂肪细胞形成的转录因子C/EBPZ及其应用 技术领域
本发明涉及动物分子遗传育种和发育生物学领域，特别是涉及一种调控脂肪细胞形成的转录因子C/EBPZ及其应用。
背景技术
经过育种工作者近百年的不懈努力，肉鸡育种已经取得了巨大进展。目前，商品代肉仔鸡的日增重和饲料转化率都达到了很高的水平。但是，由于长期育种过程中对生长性状的过度选择，腹部脂肪蓄积过多成为了当前肉鸡生产领域面临的一个重要问题。腹部脂肪过多蓄积不仅影响肉鸡生产效率，造成饲料浪费，而且造成肉鸡猝死和胴体品质下降。培育优质低脂肉鸡是控制肉鸡腹部脂肪过多蓄积的有效手段。
培育优质低脂肉鸡必须首先了解肉鸡腹部脂肪组织沉积的分子机制。腹部脂肪组织(abdominal adipose tissues)，又叫内脏脂肪组织，位于腹腔内，围绕在胃肠等脏器的周围；主要由白色脂肪细胞(white adipocyte)构成，是动物重要的能量储存库和内分泌器官。
腹部脂肪组织沉积主要发生在肉鸡生长发育阶段。脂肪细胞形成是导致脂肪组织沉积的直接原因。动物体内脂肪细胞前体主要存在于脂肪基质血管成分(adipose stromal-vascular fraction,SVF)中。目前研究者已经建立了研究鸡脂肪组织形成的有效手段。在动物水平，利用血浆极低密度脂蛋白(very low density lipoprotein,VLDL)和腹部脂肪含量双向选择等技术手段建立的高、低腹部脂肪含量肉鸡群体、以及肉鸡生产中广泛使用的商品代“快大型”肉鸡，均是适合研究鸡腹部脂肪沉积的动物 模型。全基因组关联分析、转录组学、蛋白质组学等组学技术的发展和应用，以及比较生物学的研究报道为研究鸡腹部脂肪组织沉积提供了众多有研究价值的候选基因。在细胞水平，从鸡腹部脂肪组织分离血管基质细胞(前脂肪细胞)和成熟脂肪细胞的技术；以及多种体外诱导鸡脂肪细胞形成(包括多种脂肪酸、Cocktail诱导液+油酸、Cocktail+PPARγ配体激活剂等)方法的应用，使得在体外研究鸡脂肪细胞形成具备了条件。
鸡脂肪细胞形成的研究目前已经取得了一些有价值的成果：神经多肽Y(neuropeptide Y,NPY)和BMP4等细胞外信号分子，PPARγ、C/EBPα、SREBP1、KLF2、KLF3、KLF5和KLF7等转录因子，以及gga-miR-21等miRNA在鸡脂肪细胞形成中具有重要调控作用。
CCAAT/增强子结合蛋白(CCAAT/enhancer binding proteins,C/EBPs)是一类碱性亮氨酸拉链(bZIP)转录因子家族。这个名字由史蒂文·L·麦克奈特(Steven L McNight)创造，命名的原因是第一个被确定的C/EBP蛋白具有与几个启动子CCAAT盒(CCAAT box)和一些病毒增强子的核心同源区结合的能力。在哺乳动物中一共发现了六个C/EBP家族的成员，分别为：C/EBPα、C/EBPβ、C/EBPγ、C/EBPδ、C/EBPε和DDIT3。
CCAAT盒是最早被发现能够调控真核基因表达的DNA元件之一，早期的研究报告认为C/EBPs能够结合CCAAT盒。但是近年来的研究报道显示C/EBPs并不能识别CCAAT盒，它们识别的位点是TT(G/A)CGCAA，而不是GGCCAATCT。
真正能够与GGCCAATCT结合的转录因子是CCAAT/增强子结合蛋白zeta(CCAAT/enhancer binding protein zeta,C/EBPZ)。C/EBPZ是一个特殊 的基因，严格意义上说它不属于C/EBP家族，因为它不具备典型的碱性亮氨酸拉链结构。C/EBPZ又被称为CBF、CBF-2、NOC1和HSP-CBF，最早在研究人HSP70的转录调控时被发现。
值得指出的是，有一段时间DDIT3(Gene ID:1649)曾被命名为C/EBPZ，但是它是与目前具有官方名C/EBPZ的基因(Gene ID:10153)完全不相同的一个基因，这一曾经的命名混乱给研究者在研究C/EBPZ的时候造成一些困扰，导致人们对目前具有官方名的C/EBPZ基因研究的较少，目前人们对C/EBPZ在多个生物学过程中的功能还知之甚少，还没有C/EBPZ在脂肪组织形成中的作用的研究报道。
发明内容
本发明的目的是提供一种调控脂肪细胞形成的转录因子C/EBPZ及其应用，以解决上述现有技术存在的问题。
为实现上述目的，本发明提供了如下方案：
本发明提供一种调控鸡脂肪细胞形成的转录因子C/EBPZ，所述转录因子C/EBPZ的编码基因序列如SEQ ID NO.1所示。
本发明还提供一种所述转录因子C/EBPZ在制备一种制剂或组合物中的用途，所述制剂或组合物用于调控鸡脂肪细胞形成。
进一步地，所述调控鸡脂肪细胞形成是指调控鸡脂肪细胞的分化和增殖。
进一步地，所述调控鸡脂肪细胞的分化是指过表达转录因子C/EBPZ抑制鸡脂肪细胞的分化。
进一步地，所述调控鸡脂肪细胞的增殖是指过表达转录因子C/EBPZ 促进鸡脂肪细胞的增殖。
进一步地，所述调控鸡脂肪细胞形成的基因选自PPARγ、FASN、C/EBPα、LPL、FABP4、GATA2或其组合。
进一步地，通过鸡脂肪细胞中转录因子C/EBPZ的过表达调控所述基因的启动子活性和mRNA表达。
本发明还提供一种所述转录因子C/EBPZ在筛选肉鸡中的用途。
进一步地，所述筛选具体为：检测肉鸡腹部脂肪组织中C/EBPZ基因的mRNA表达水平，检测C/EBPZ基因表达所用引物对的核苷酸序列如SEQ ID NO.3和SEQ ID NO.4所示。
本发明提供一种重组质粒pCMV-HA-C/EBPZ，所述重组质粒pCMV-HA-C/EBPZ包括所述转录因子C/EBPZ，所述重组质粒pCMV-HA-C/EBPZ的核苷酸序列如SEQ ID NO.2所示。
本发明公开了以下技术效果：
本发明公开一种转录因子C/EBPZ在调控鸡脂肪细胞形成中应用，在动物水平上，利用real-time PCR技术在鸡腹部脂肪组织中检测C/EBPZ基因mRNA表达水平，验证了该基因在高低脂系肉鸡腹部脂肪组织中表达水平存在显著差异。在细胞水平上，构建该转录因子的过表达载体，验证了C/EBPZ转录因子调控鸡前脂肪细胞分化和对鸡前脂肪细胞增殖的调控作用；本发明进一步揭示了转录因子C/EBPZ在鸡脂肪组织形成中的作用机制，该转录因子通过调控PPARγ、FASN、C/EBPα、LPL、FABP4、GATA2和KLF2等或其组合的表达，进而影响鸡前脂肪的增殖和分化。本发明提供了C/EBPZ转录因子在调控鸡脂肪细胞形成中的新用途，在鸡遗传育种和动物营养领 域具有实用价值。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1为C/EBPZ过表达载体(pCMV-HA-C/EBPZ)和空载体(pCMV-HA)的质粒图谱；
图2为C/EBPZ基因在肉鸡多种组织中的表达模式；其中：1：腹部脂肪，2：脑，3：十二指肠，4：肌胃，5：心脏，6：回肠，7：空肠，8：胃，9：腿肌，10：肝，12：胸肌，13：腺胃，14：脾，15：睾丸；
图3为C/EBPZ基因在肉鸡腹部脂肪组织生长发育中的表达模式(n＝106)；其中A是高低系肉鸡腹部脂肪组织C/EBPZ表达水平数据核密度图，B是1-14周龄高低脂系肉鸡腹部脂肪C/EBPZ表达水平图；
图4为C/EBPZ过表达载体的质粒表达蛋白验证；
图5为过表达C/EBPZ对鸡前脂肪细胞增殖影响图；
图6为为过表达C/EBPZ抑制鸡前脂肪细胞分化图；
图7为过表达C/EBPZ调控PPARγ、FASN、C/EBPα、LPL、FABP4、GATA2启动子活性图；
图8为C/EBPZ过表达48h后鸡脂肪细胞中转录组基因集富集分析；其中A是RNA-seq分析显示的过表达C/EBPZ与对照组相比的差异基因数目图；B是对RNA-seq结果数据进行基因集富集分析图；
图9为验证RNA-seq结果所做的过表达C/EBPZ对鸡前脂肪细胞中内源性脂肪细胞形成标志基因表达的影响real-time PCR结果图。
具体实施方式
现详细说明本发明的多种示例性实施方式，该详细说明不应认为是对本发明的限制，而应理解为是对本发明的某些方面、特性和实施方案的更详细的描述。
应理解本发明中所述的术语仅仅是为描述特别的实施方式，并非用于限制本发明。另外，对于本发明中的数值范围，应理解为还具体公开了该范围的上限和下限之间的每个中间值。在任何陈述值或陈述范围内的中间值以及任何其他陈述值或在所述范围内的中间值之间的每个较小的范围也包括在本发明内。这些较小范围的上限和下限可独立地包括或排除在范围内。
除非另有说明，否则本文使用的所有技术和科学术语具有本发明所述领域的常规技术人员通常理解的相同含义。虽然本发明仅描述了优选的方法和材料，但是在本发明的实施或测试中也可以使用与本文所述相似或等同的任何方法和材料。本说明书中提到的所有文献通过引用并入，用以公开和描述与所述文献相关的方法和/或材料。在与任何并入的文献冲突时，以本说明书的内容为准。
在不背离本发明的范围或精神的情况下，可对本发明说明书的具体实施方式做多种改进和变化，这对本领域技术人员而言是显而易见的。由本发明的说明书得到的其他实施方式对技术人员而言是显而易见得的。本申请说明书和实施例仅是示例性的。
关于本文中所使用的“包含”、“包括”、“具有”、“含有”等等，均为开放性的用语，即意指包含但不限于。
下列实施例中使用的实验方法如无特殊说明，均为常规方法；下列实施例中所使用的材料、试剂等，如无特殊说明，均可从商业途径得到。
实施例1鸡C/EBPZ基因的克隆和mRNA表达水平的检测
1.组织取样
以东北农业大学肉鸡高、低脂双向选择系(Northeast Agricultural University broiler lines divergently selected for abdominal fat content,NEAUHLF)第十四世代1-12周龄肉鸡作为实验材料，从1到12周龄，每周屠宰十只公鸡(高低脂各5只)。在每个周龄，屠宰后均收集腹部脂肪垫。在7周龄，其他15个组织，包括肝，十二指肠，空肠，回肠，胸，腿部肌肉，肌胃周围脂肪，心脏，脾，肾，胰腺，腺胃，大脑，睾丸也被收集。所有收集的组织马上用液氮冷冻，并储存到-80℃冰箱中备用。
2、RNA提取
1)取100mg收集的鸡组织样本，在液氮中研磨后，加入1mL Trizol试剂(Invitrogen)。
2)将上述标本转移到1.5mL干净EP管中，室温静置5min，加入0.2mL的氯仿，剧烈震荡15s混匀，室温静置2min。
3)12000g，4℃，离心15min，将上层水相转移至一个新的干净的EP管中，加入0.5mL异丙醇，室温静置10min。
4)12000g，4℃，离心10min，弃掉上清。
5)在上述EP管中，加入1mL 75％的乙醇振荡重悬RNA，7500g，4℃ 离心5min。
6)弃去上清，在空气中静置5-10min干燥RNA。
7)用40μL RNase free水重悬RNA，-80℃保存备用。
3、反转录
以提取的总RNA为模板，利用ImProm-II reverse transcriptase Kit(Promega)进行反转录，反应体系I：总RNA 1.0μg，oligod T引物(2.5μM)0.5μL，加RNase free ddH 2O补足5μL。将上述反应体系I在70℃处理5min，然后在冰上放置5min，以破坏RNA的二级结构，之后配置反应体系II，如下：
Figure PCTCN2021124592-appb-000001
反转录条件如下：
Figure PCTCN2021124592-appb-000002
4、鸡C/EBPZ基因的克隆
用引物C/EBPZ克隆引物(CEBPZ CF：GTCGACCATGGCGGCGCTCGGGGAGT和CEBPZ CR：AGATCTTCATCTTTTTGATTTCTTGCCTC)进行PCR扩增。
反应体系如下：
Figure PCTCN2021124592-appb-000003
PCR扩增条件为：94℃预变性7min；94℃变性30s，62℃退火30s，72℃延伸2min，共35个循环，72℃终延伸7min，4℃终止反应。
将PCR产物进行琼脂糖凝胶电泳，回收目的条带。将目的基因连接在pMD-18T载体(Takara)上，连接产物转入大肠杆菌DH5α，AMP抗性筛选培养，挑取单克隆，将单克隆接种于LB液体培养基，提取质粒，将质粒进行测序，测得插入pMD-18T中的C/EBPZ核苷酸序列如SEQ ID NO.1所示，将得到的阳性重组质粒标记为pMD-18T-C/EBPZ。
3、鸡C/EBPZ过表达载体质粒的构建
分别以pMD-18T-C/EBPZ重组质粒和pCMV-HA载体为酶切底物，利用SalI和BglⅡ两个内切酶进行双酶切，体系如下所示：
Figure PCTCN2021124592-appb-000004
Figure PCTCN2021124592-appb-000005
酶切条件为：37℃酶切1h。
酶切产物经1％琼脂糖凝胶电泳鉴定并用AXYGEN凝胶回收纯化试剂盒纯化回收，利用T4 DNA连接酶，将目的基因片段和骨架载体片段连接到一起，构建pCMV-HA-C/EBPZ载体，连接产物转入大肠杆菌DH5α，AMP抗性筛选培养，挑去单克隆，将单克隆接种于LB液体培养基，提取质粒，利用SalI和BglⅡ进行双酶切和测序鉴定，得到的阳性重组质粒标记为pCMV-HA-C/EBPZ，重组质粒的序列如SEQ ID NO.2所示。C/EBPZ过表达载体(pCMV-HA-C/EBPZ)载体和空载体(pCMV-HA)的质粒图谱如图1所示。
5、Real-time PCR检测C/EBPZ的表达水平
Real-time PCR利用SYBR Premix Ex Taq试剂盒(Takara公司)和ABI Prism 7500 sequence detection system(Applied Biosystems公司)完成，反应采用20μL体系，反应体系在冰上配置，体系包括：cDNA 2μL、2×SYBR Premix Ex Taq 10μL、PCR Forward(Reverse)Primer(10μmol/L)各0.4μL、50×Rox Reference DyeⅡ 0.4μL、双蒸水6.8μL；反应条件为95℃预变性5s，而后进行40个循环，每个循环包括95℃5s和60℃34s；40个循环完成后进行融解曲线(dissociation curves)检测。分析C/EBPZ表达所用的引物见SEQ ID NO.3和SEQ ID NO.4。结果表明，C/EBPZ在鸡多种组织中广泛表达(图2)，并且在低脂系肉鸡腹部脂肪组织中C/EBPZ的相对表达水平(C/EBPZ/β-ACTIN)显著高于高脂系肉鸡(图3A，P<0.01),并 且在第7和第9周龄时低脂系肉鸡腹部脂肪组织C/EBPZ mRNA相对表达水平显著高于高脂系肉鸡(图3B，P<0.05)。提示C/EBPZ mRNA相对表达水平(C/EBPZ/β-ACTIN)可以作为腹部脂肪含量的一个分子标志。
实施例2过表达C/EBPZ对鸡前脂肪细胞形成的影响
1、鸡前脂肪细胞分离和培养
1)从12日龄AA肉鸡中取腹部脂肪组织(3-5克)，用PBS清洗2遍后，用2mg/mL的I型胶原酶(Sigma)37℃消化1小时，每隔10分钟上下颠倒混匀一次。
2)让消化后的组织液通过100微米和600微米的滤网，除去未消化的组织块。
3)收集过滤后的组织消化液200g离心10分钟。
4)静置10分钟，让细胞分层，下层细胞再次经过红细胞裂解液处理后，200g离心10分钟，获得的鸡腹部脂肪组织来源的血管基质(SVF)细胞，作为鸡前脂肪细胞使用。
5)分离的鸡前脂肪细胞用全培养基(DMEM/F12+10％FBS+1％K)悬浮，以密度1×10 5cells/cm 2接种到细胞培养瓶中，保然后在37℃，5％CO 2的条件下培养。
2、C/EBPZ过表达载体在鸡前脂肪细胞中表达蛋白的验证
将生长状态良好的鸡前脂肪细胞接种于6孔细胞培养板中，接种密度为1×10 5个/孔，传代24h后，大约细胞70-90％汇合，按照Fugene HD(Promega)说明书分别将空表达载体质粒pCMV-HA和C/EBPZ过表达质粒pCMV-HA-C/EBPZ分别转染到6孔板培养的鸡前脂肪细胞中，每孔转染2.0 μg质粒，转染后48h回收细胞，利用western blotting验证pCMV-HA-C/EBPZ载体能否表达融合蛋白，结果显示转染pCMV-HA-C/EBPZ的细胞中成功表达出大小约为130kD的蛋白，与预测的鸡C/EBPZ融合蛋白大小相近，而转染pCMV-HA的细胞没有表达出类似大小的HA融合蛋白如图4所示，表明在鸡前脂肪细胞中转染pCMV-HA-C/EBPZ可以过表达鸡C/EBPZ的蛋白质。
3、过表达转录因子C/EBPZ对鸡前脂肪细胞增殖的影响
将生长状态良好的鸡前脂肪细胞接种于6孔细胞培养板中，接种密度为1×10 5个/孔，传代24h之后，大约细胞70-90％汇合，按照Fugene HD(promega)说明书分别将质粒pCMV-HA和pCMV-HA-C/EBPZ转染到不同孔的鸡前脂肪细胞细胞中，生长24h之后，利用胰蛋白酶将贴壁细胞消化下来，以每孔5000个细胞的浓度接种到96孔细胞培养板中，接种后24h、48h、72h、96h和120h时间点，利用
Figure PCTCN2021124592-appb-000006
发光法细胞活力检测试剂盒(Promega)检测细胞增殖数目，结果如图5所示，在96h和120h时间点，过表达C/EBPZ的前脂肪细胞与对照组相比增殖能力显著增强(P<0.05，不配对双尾T检验)，过表达C/EBPZ促进鸡前脂肪细胞增殖。
4、过表达转录因子C/EBPZ对鸡前脂肪细胞分化的影响
将生长状态良好的鸡前脂肪细胞接种于6孔细胞培养板中，接种密度为1×10 5个/孔，传代24h后，大约细胞70-90％汇合，采用Fugene HD转染试剂(Promega)按照说明书分别将质粒pCMV-HA和pCMV-HA-C/EBPZ转染到不同孔的鸡前脂肪细胞细胞中，转染后大约48h，当细胞培养板中的细胞完全长满(细胞100％汇合)后，利用160nM的油酸钠(Sigma)对前 脂肪细胞进行诱导脂肪细胞分化。诱导分化48小时后，弃去培养基，用PBS洗3次，在10％甲醛固定10分钟。然后用蒸馏水冲洗固定液，用0.5％的油红O染色30分钟。弃去多余的染色液，用PBS冲洗两次。在肉眼和倒置显微镜下观察细胞形态。结果如图6所示，在48h时间点，转染pCMV-HA-C/EBPZ的鸡前脂肪细胞与对照组细胞相比，脂肪含量明显减少，表明过表达C/EBPZ抑制鸡前脂肪细胞的分化。
5、在鸡前脂肪细胞中过表达C/EBPZ对PPARγ、C/EBPα、FASN、LPL、FABP4和GATA2启动子活性的影响
将生长状态良好的鸡前脂肪细胞接种于12孔细胞培养板中，接种密度为5×10 4个/孔，24h之后，按照Fugene HD(Promega)说明书将各组质粒转染到鸡前脂肪细胞中，分组如下所示：
Figure PCTCN2021124592-appb-000007
Figure PCTCN2021124592-appb-000008
每组重复三次，48h之后收细胞，按照Promega公司的
Figure PCTCN2021124592-appb-000009
Luciferase Assay System说明书进行荧光活性测定，结果如图7所示，与转染空载体的对照组相比，过表达C/EBPZ在鸡前脂肪细胞对C/EBPα和FASN启动子活性无显著影响(P>0.05)，过表达C/EBPZ在鸡前脂肪细胞抑制PPARγ和LPL启动子活性(P<0.05,不配对双尾T检验)，促进脂肪细胞分化抑制因子GATA2的启动子活性,过表达C/EBPZ促进FABP4启动子活性。上述基因均为脂肪细胞分化调控基因，表明过表达C/EBPZ通过调控上述基因的转录活性调控鸡前脂肪细胞分化。
6、RNA-seq技术分析过表达C/EBPZ对鸡前脂肪细胞中RNA表达模式的影响
将生长状态良好的鸡前脂肪细胞以1×10 5cells/cm 2的密度接种到6孔板密度。接种12小时后，细胞大约长到60-80％汇合，按照Fugene HD(Promega)转染试剂按照说明书转染pCMV-HA-C/EBPZ或空载体(pCMV-HA)质粒转染到细胞中，转染后48h后，收集细胞，委托天津诺禾致源生物信息科技有限公司进行转录组RNA-seq分析。RNA-seq结果图8A所示，与对照组相比，过表达C/EBPZ后，448个基因上调表达，354个基因下调表达；如图8B所示，基因集富集分析(gene set enrichment analysis,GSEA)显示鸡前脂肪细胞中过表达C/EBPZ后，脂肪组织发育基因(ADIPOSE_TISSUE_DEVELOPMENT，GO:0060612))表达显著上调(P<0.01)，表明过表达C/EBPZ参与脂肪细胞形成的调控。
7、Real time PCR分析过表达C/EBPZ对鸡前脂肪细胞内源性基因表 达的影响
将生长状态良好的鸡前脂肪细胞以1×10 5cells/cm 2的密度接种到6孔板密度。接种12小时后，细胞大约长到60-80％汇合，按照Fugene HD(Promega)转染试剂按照说明书转染pCMV-HA-C/EBPZ或空载体(pCMV-HA)质粒转染到细胞中。转染后48h后，收集细胞。
用TRIzol试剂收集贴壁的鸡前脂肪细胞并从中提取总RNA，用Nano drop 2000(Thermo scientific)测定提取的RNA浓度，取1μg RNA使用Promega-Improm II(Promega)反转录试剂套装进行反转录成cDNA，反转录条件为：25.0℃5min,42.0℃60min,70.0℃15min,5.0℃保温。然后取1微升cDNA产物采用10μL体系,体系包括：2×QuantiNova SYBR Green RT-PCR Master Mix 5μL、PCR Forward(Reverse)Primer(10μmol/L)各0.2μL、cDNA产物1μL、双蒸水3.6μL。使用QIAGEN qRT-PCR仪器系统(QIAGEN,Hilden,Germany)进行real time PCR分析，程序设定为：95.0℃进行5min,然后40个循环：95.0℃进行10s，56.0℃进行30s,72.0℃进行40s加解离曲线，使用Rotor-Gene Q Series Software2.3.1软件进行数据分析，结果如图9所示，过表达C/EBPZ抑制鸡前脂肪细胞中内源性脂肪细胞分化标志基因FASN表达的表达水平(P<0.05,不配对双尾T检验),促进脂肪细胞分化抑制基因KLF2的表达水平(P<0.05,不配对双尾T检验)。此外，过表达C/EBPZ促进应激相关基因HSPB8和激素诱导的脂肪分解激活基因TNFAIP6和GIT1的表达(P<0.05,不配对双尾T检验)，对PPARγ、C/EBPα、KLF7、ACADL、GATA2和LPL内源性表达的影响没有达到显著水平(P>0.05,不配对双尾T检验)，该结果与RNA-seq分析结果一致，表明 过表达C/EBPZ调控鸡脂肪组织形成。
所用的引物如下表1所示：
表1
Figure PCTCN2021124592-appb-000010
以上所述的实施例仅是对本发明的优选方式进行描述，并非对本发明的范围进行限定，在不脱离本发明设计精神的前提下，本领域普通技术人员对本发明的技术方案做出的各种变形和改进，均应落入本发明权利要求书确定的保护范围内。

Claims (10)

  1. 一种调控鸡脂肪细胞形成的转录因子C/EBPZ，其特征在于，所述转录因子C/EBPZ的编码基因序列如SEQ ID NO.1所示。
  2. 一种权利要求1所述转录因子C/EBPZ在制备一种制剂或组合物中的用途，其特征在于，所述制剂或组合物用于调控鸡脂肪细胞形成。
  3. 根据权利要求2所述的用途，其特征在于，所述调控鸡脂肪细胞形成是指调控鸡脂肪细胞的分化和增殖。
  4. 根据权利要求3所述的用途，其特征在于，所述调控鸡脂肪细胞的分化是指过表达转录因子C/EBPZ抑制鸡脂肪细胞的分化。
  5. 根据权利要求3所述的用途，其特征在于，所述调控鸡脂肪细胞的增殖是指过表达转录因子C/EBPZ促进鸡脂肪细胞的增殖。
  6. 根据权利要求2所述的用途，其特征在于，所述调控鸡脂肪细胞形成的基因选自PPARγ、FASN、C/EBPα、LPL、FABP4、GATA2或其组合。
  7. 根据权利要求6所述的用途，其特征在于，通过鸡脂肪细胞中转录因子C/EBPZ的过表达调控所述基因的启动子活性和mRNA表达。
  8. 一种权利要求1所述转录因子C/EBPZ在筛选肉鸡中的用途。
  9. 根据权利要求8所述的用途，其特征在于，所述筛选具体为：检测肉鸡腹部脂肪组织中C/EBPZ基因的mRNA表达水平，检测C/EBPZ基因表达所用引物对的核苷酸序列如SEQ ID NO.3和SEQ ID NO.4所示。
  10. 一种重组质粒pCMV-HA-C/EBPZ，其特征在于，所述重组质粒pCMV-HA-C/EBPZ包括权利要求1所述转录因子C/EBPZ，所述重组质粒pCMV-HA-C/EBPZ的核苷酸序列如SEQ ID NO.2所示。
PCT/CN2021/124592 2020-12-11 2021-10-19 一种调控脂肪细胞形成的转录因子c/ebpz及其应用 WO2022121509A1 (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
ZA2023/03608A ZA202303608B (en) 2020-12-11 2023-03-15 Transcription factor c/ebpz for regulating adipocyte formation and application thereof

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202011458514.3 2020-12-11
CN202011458514.3A CN112481271B (zh) 2020-12-11 2020-12-11 一种调控脂肪细胞形成的转录因子c/ebpz及其应用

Publications (1)

Publication Number Publication Date
WO2022121509A1 true WO2022121509A1 (zh) 2022-06-16

Family

ID=74917730

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2021/124592 WO2022121509A1 (zh) 2020-12-11 2021-10-19 一种调控脂肪细胞形成的转录因子c/ebpz及其应用

Country Status (3)

Country Link
CN (1) CN112481271B (zh)
WO (1) WO2022121509A1 (zh)
ZA (1) ZA202303608B (zh)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111500590B (zh) * 2020-05-11 2022-09-16 石河子大学 一种调控鸡脂肪细胞形成的转录因子gata2的应用
CN112481271B (zh) * 2020-12-11 2024-04-30 石河子大学 一种调控脂肪细胞形成的转录因子c/ebpz及其应用
CN113881678B (zh) * 2021-11-01 2024-04-12 石河子大学 一种c/ebpz基因启动子及其应用

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105087584A (zh) * 2015-08-12 2015-11-25 中国农业科学院北京畜牧兽医研究所 一种与鸡腹脂沉积相关的miRNA及其应用
CN110004153A (zh) * 2019-04-16 2019-07-12 常熟理工学院 鸡重组CEBPγ蛋白、其编码DNA序列及应用
CN111500590A (zh) * 2020-05-11 2020-08-07 石河子大学 一种调控鸡脂肪细胞形成的转录因子gata2的应用
CN112481271A (zh) * 2020-12-11 2021-03-12 石河子大学 一种调控脂肪细胞形成的转录因子c/ebpz及其应用

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011028819A1 (en) * 2009-09-01 2011-03-10 The Trustees Of Columbia University In The City Of New York Synergistic transcription modules and uses thereof
US20170002319A1 (en) * 2015-05-13 2017-01-05 Whitehead Institute For Biomedical Research Master Transcription Factors Identification and Use Thereof

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105087584A (zh) * 2015-08-12 2015-11-25 中国农业科学院北京畜牧兽医研究所 一种与鸡腹脂沉积相关的miRNA及其应用
CN110004153A (zh) * 2019-04-16 2019-07-12 常熟理工学院 鸡重组CEBPγ蛋白、其编码DNA序列及应用
CN111500590A (zh) * 2020-05-11 2020-08-07 石河子大学 一种调控鸡脂肪细胞形成的转录因子gata2的应用
CN112481271A (zh) * 2020-12-11 2021-03-12 石河子大学 一种调控脂肪细胞形成的转录因子c/ebpz及其应用

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
BYERLY MARDI S., SIMON JEAN, COGBURN LARRY A., LE BIHAN-DUVAL ELISABETH, DUCLOS MICHEL J., AGGREY SAMUEL E., PORTER TOM E.: "Transcriptional profiling of hypothalamus during development of adiposity in genetically selected fat and lean chickens", PHYSIOLOGICAL GENOMICS, vol. 42, no. 2, 1 July 2010 (2010-07-01), US , pages 157 - 167, XP055941963, ISSN: 1094-8341, DOI: 10.1152/physiolgenomics.00029.2010 *
DATABASE Nucleotide 12 January 2005 (2005-01-12), ANONYMOUS: "Gallus gallus mRNA for hypothetical protein, clone 24e3", XP055941973, retrieved from Genbank Database accession no. AJ720727 *

Also Published As

Publication number Publication date
CN112481271B (zh) 2024-04-30
CN112481271A (zh) 2021-03-12
ZA202303608B (en) 2023-04-26

Similar Documents

Publication Publication Date Title
WO2022121509A1 (zh) 一种调控脂肪细胞形成的转录因子c/ebpz及其应用
JP6903355B2 (ja) 多能性幹細胞を所望の細胞型へ分化する方法
Caputo et al. The Isl1/Ldb1 complex orchestrates genome-wide chromatin organization to instruct differentiation of multipotent cardiac progenitors
Mowel et al. Group 1 innate lymphoid cell lineage identity is determined by a cis-regulatory element marked by a long non-coding RNA
CN111154763B (zh) 长链非编码RNA lncMGPF在调控猪肌肉发育功能中的应用
CN110628766B (zh) 与羊骨骼肌发育相关的lncRNA编码基因及其应用
WO2022135279A1 (zh) Myog基因作为靶点在制备治疗心肌细胞凋亡相关的心血管疾病的药物中的应用
CN115029351B (zh) 一种shRNA或BACH1缺失巨噬细胞来源的EVs在制备治疗高血压的药物中的应用
Yin et al. Identification and expression of the target gene emx2 of miR-26a and miR-26b in Paralichthys olivaceus
Wang et al. RIP-Seq of EZH2 Identifies TCONS-00036665 as a Regulator of Myogenesis in Pigs
Deng et al. The transcriptomes from two adipocyte progenitor cell types provide insight into the differential functions of MSTN
CN114480672A (zh) 一种通过miR-145筛选高产肉率碱地黑牛的方法
CN110157736B (zh) 一种促进山羊毛囊干细胞增殖的方法
Stahlhut et al. Lentiviral vector system for coordinated constitutive and drug controlled tetracycline-regulated gene co-expression
Wang et al. Multiple alternative splicing and differential expression of actinin-associated LIM protein (ALP) during porcine skeletal muscle development in vitro and in vivo
Ren et al. Identification of a Yorkie homolog from Litopenaeus vannamei as a negative regulator in anti-WSSV immune response
CN107119072B (zh) 一种过表达zeb2基因质粒及其构建方法和应用
CN113230268B (zh) 一种非编码小rna基因、载体和细胞在制备抑制乳腺癌的药物中的用途
CN117187249B (zh) 一种牦牛Lnc-MEG8基因及其应用
CN113957157B (zh) 一种与蛋鸡肝脏脂代谢相关的circRNA及其应用
CN117248036A (zh) 一种与鸡脂肪沉积相关的miRNA家族及其应用
Wang et al. Retinoblastoma 1 (RB1) modulates the proliferation of chicken preadipocytes
Wang et al. The retinoblastoma 1 gene (RB1) modulates the proliferation of chicken preadipocytes
CN115948538A (zh) Dusp13在治疗心肌细胞凋亡相关的心血管疾病中的应用
EP3218478A1 (en) Predicting productivity in early cell line development

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21902216

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 21902216

Country of ref document: EP

Kind code of ref document: A1