CN112481271B - 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 PDFInfo
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Abstract
本发明公开了一种调控脂肪细胞形成的转录因子C/EBPZ及其应用,属于动物分子遗传学和发育生物学领域,本发明公开了转录因子C/EBPZ调控鸡脂肪细胞形成的用途;本发明利用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。本发明构建了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的分化和增殖;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。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。
Description
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(adiposestromal-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 LMcNight)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(CCAATbox)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 Universitybroiler 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 ddH2O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:
反转录条件如下:
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:
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SEQID 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:
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 Prism7500sequence 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 DyeⅡ0.4μL、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 IDNO.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1×105cells/cm2接种到细胞培养瓶中,保然后在37℃,5%CO2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1×105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2.0μg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1×105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1×105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5×104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:
每组重复三次,48h之后收细胞,按照Promega公司的LuciferaseAssay 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1×105cells/cm2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1×105cells/cm2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 SYBRGreen 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realtime 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
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。
序列表
<110> 石河子大学
<120> 一种调控脂肪细胞形成的转录因子C/EBPZ及其应用
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 3244
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
gtcgaccatg gcggcgctcg gggagtgggg cgtgcggcgg cggccggagg aggcggatgg 60
ctacgaactg gatgaagatg atgatgatgg tgatggtgat gatggtggag atgccgatgg 120
gttcacgctg gatgaggtgc tccgcctcgg cggcaccaag caagacttcc tcatgctgtg 180
cggcgtggac gaggcggagg agatggtgga cggcggcaag aaaggtgcca tcgacgatct 240
gaaggagggg gagctggagg ccttcatcgg ctccttgggg cttggcaagt atgcaaaagg 300
ctgcctggac gaggaggagg aggaggagga gaacgaggag aagaagggca gcaaagagaa 360
agagaagatc ccaaagaagg agaaaagcaa gaaggagaaa aacaaggaga aaagcaagaa 420
ggaaacagtt gctccttcat cagaagggaa aaaggaaaaa aaaaccgaag agcaagtgag 480
tagcagaaaa gacagcaaga agaagctagc tggcggtggt ccacgccagc agcagtcggt 540
ccagaaggag aggctggaag aggacttcac atttcgtgct agggaggtga tactgatcaa 600
acctggcggc aagtggtacg acctcgagta caccggtgag acttctgctg agccgcagga 660
tccagccctc ctgtcccagt acaaggcctt ggcccaaaag ctgtaccagc acgagacaga 720
gctgtacaag agcaagacag agtatcagaa gggagcctcg tcggcatgga tgaaaaccgt 780
tgtgtcgtcg ggtaccctcg ctgacaggat ggcagccatg accctcctca tccaggacag 840
cgctgttcac agtctccact ttgttgagaa cctggtgaac ctcgtaaaga aaaagggcag 900
caggcaacag agcctcatgg ctctggatac tttcagagag cttctccttt ctgatctctt 960
accagatact cgcaagctgt ggtctttctc acagcgtcca cttaacaaca tagagaaaat 1020
gtcgagtggc aacagagatt ccagggacag acggctgata ctatggtact ttgagcacca 1080
gctgaagctg ttggtggcag agtttgtgca gaccttggaa gcactgagcc acgattccct 1140
gacagcaaca aaaacccgag cgctggcagc tgcccacgag cttctctgca acaagcctga 1200
gcaggagaaa tttctgcttg tccagctggt aaataaactg ggagaccctc agaacaaaat 1260
agccaccaaa gcgtcttatc ttctagagac tttacttcac aagcatccaa atatgaaagg 1320
agtggtgtgc agtgaggtgg agagactttt gtaccgcaca aacatcagtg caaagactca 1380
gtattacgcc atgtgctttt taaatcagat agtcctcagt catgaagaaa caccgttggc 1440
caacaagctg ataactttgt acttctgttt ttttcggaac tgcattaaga aaaaagatgt 1500
cgaatccaaa atgctcagcg ctcttcttag cggggttaac agagcttacc cttatgctga 1560
gattggtgat gagaaagtga aagaacaaat gaacaccttg tttaaagttc tacaccttgt 1620
gaacttcagc accagcgtcc aggccctgat gttgctgttc caggttatgg actcccagca 1680
gactgtatcc gataggtact acgcagcact gtataagaag cttctagatc ccggtttagc 1740
aacctgctca aagccatcca tgtttcttaa tcttgtctat aaatctctga aggcagacgt 1800
ggtgttacgg cgagtgaagg ccttcgtgaa gaggttgctt caggtcacta ctggacagac 1860
accacccttc atttgtggaa ccctgtacct tctgtctgag cttctgaaag taaaaccagg 1920
cctgagggtc cagttacagg atcacgtgga gtctgatgaa gaagagtgct ttaaggatca 1980
agaagagact gaagagaacg aggaaacatt tgtggatgca gacaaagtag aaagggaaga 2040
gaggagcgca acagaaaatt ctgctaaaag aaataatctg agttcagcag cctcatgggt 2100
gcatcatgag aatatgggag ggagaaagaa tggggtttcc tacgatcctt tgcaccgaag 2160
tcctttatac tgtggtgctg aaagtacaag cctttgggaa ctgaagaagc tttctgaaca 2220
ttttcatcca tctgtggctc tctttgcaaa aaccattcta gagggaaatc atattcagta 2280
ctccggtgac cctttgcaag atttcacatt aatgagattc ttggatcgct ttgtgtaccg 2340
aaatcccaaa ctccacaaag gcaaagagaa caccagcagt gtggtaatgc agcggaagaa 2400
gaagcagttt atgacggata cacaaaatct ggcagtcaat agtaaggaat tccttgccag 2460
agatgaaagc aaaatcccag tggatgaatt attttttcac agattctata caaagtttga 2520
taaaacgcga gagaagcaca ggcgtcagga ggatgaagaa agcgtggaag atgtggacga 2580
tgatgagttt gaaagagcac tggatacatt tgaagctgat ggtgctgttg gtgttggcca 2640
ggatgacctt gattttgctg gtaatataaa aaagaaaagc aacgggaata agaaaagcaa 2700
aagaagtgag gaatccggtg ctgactggga ggattctggc gatgaagaat tcagtgacct 2760
ggatgatgag gaagtctcct taggaagcat ggaggaagat gactttgaag aggatatgga 2820
tgaggaagga ggtgtattta tggacgtgtc tgatgatggt aatggcccag accttaacaa 2880
tgacaatcaa tcgaagtctg tcagtaaaaa gaccaagaga aagaaagata tgaattttat 2940
gggatcacat gaaggatcca gccaaggaaa gaagagaaaa ctcaaagatg ccaaaatact 3000
ggcagctgct gaagagattg gctacctgct ggatgaaaat gctgggtcca agtttgacaa 3060
tattggaatg aatgccatgg ctaacagaga taatgcaaat atcaaacaga tcaagtggga 3120
attagagcgc gacaggtggc ttcacaacag ggatgtgaaa agcatcatca aaaggaaaaa 3180
acagctcagg cacacggggc tgaaaaagaa gtacagaggc aagaaatcaa aaagatgaag 3240
atct 3244
<210> 2
<211> 7012
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
gagttcgagc ttgcatgcct gcaggtcgtt acataactta cggtaaatgg cccgcctggc 60
tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg 120
ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg 180
gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa 240
tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac 300
atctacgtat tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg 360
cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg 420
agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca 480
ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctcgttta 540
gtgaaccgtc agatcgcctg gagacgccat ccacgctgtt ttgacctcca tagaagacac 600
cgggaccgat ccagcctccg gactctagag gatccggtac tagaggaact gaaaaaccag 660
aaagttaact ggtaagttta gtctttttgt cttttatttc aggtcccgga tccggtggtg 720
gtgcaaatca aagaactgct cctcagtgga tgttgccttt acttctaggc ctgtacggaa 780
gtgttacttc tgctctaaaa gctgcggaat tgtacccgcg ggcccaccat gtacccatac 840
gatgttccag attacgctct tatggccatg gaggcccgaa ttcggtcgac catggcggcg 900
ctcggggagt ggggcgtgcg gcggcggccg gaggaggcgg atggctacga actggatgaa 960
gatgatgatg atggtgatgg tgatgatggt ggagatgccg atgggttcac gctggatgag 1020
gtgctccgcc tcggcggcac caagcaagac ttcctcatgc tgtgcggcgt ggacgaggcg 1080
gaggagatgg tggacggcgg caagaaaggt gccatcgacg atctgaagga gggggagctg 1140
gaggccttca tcggctcctt ggggcttggc aagtatgcaa aaggctgcct ggacgaggag 1200
gaggaggagg aggagaacga ggagaagaag ggcagcaaag agaaagagaa gatcccaaag 1260
aaggagaaaa gcaagaagga gaaaaacaag gagaaaagca agaaggaaac agttgctcct 1320
tcatcagaag ggaaaaagga aaaaaaaacc gaagagcaag tgagtagcag aaaagacagc 1380
aagaagaagc tagctggcgg tggtccacgc cagcagcagt cggtccagaa ggagaggctg 1440
gaagaggact tcacatttcg tgctagggag gtgatactga tcaaacctgg cggcaagtgg 1500
tacgacctcg agtacaccgg tgagacttct gctgagccgc aggatccagc cctcctgtcc 1560
cagtacaagg ccttggccca aaagctgtac cagcacgaga cagagctgta caagagcaag 1620
acagagtatc agaagggagc ctcgtcggca tggatgaaaa ccgttgtgtc gtcgggtacc 1680
ctcgctgaca ggatggcagc catgaccctc ctcatccagg acagcgctgt tcacagtctc 1740
cactttgttg agaacctggt gaacctcgta aagaaaaagg gcagcaggca acagagcctc 1800
atggctctgg atactttcag agagcttctc ctttctgatc tcttaccaga tactcgcaag 1860
ctgtggtctt tctcacagcg tccacttaac aacatagaga aaatgtcgag tggcaacaga 1920
gattccaggg acagacggct gatactatgg tactttgagc accagctgaa gctgttggtg 1980
gcagagtttg tgcagacctt ggaagcactg agccacgatt ccctgacagc aacaaaaacc 2040
cgagcgctgg cagctgccca cgagcttctc tgcaacaagc ctgagcagga gaaatttctg 2100
cttgtccagc tggtaaataa actgggagac cctcagaaca aaatagccac caaagcgtct 2160
tatcttctag agactttact tcacaagcat ccaaatatga aaggagtggt gtgcagtgag 2220
gtggagagac ttttgtaccg cacaaacatc agtgcaaaga ctcagtatta cgccatgtgc 2280
tttttaaatc agatagtcct cagtcatgaa gaaacaccgt tggccaacaa gctgataact 2340
ttgtacttct gtttttttcg gaactgcatt aagaaaaaag atgtcgaatc caaaatgctc 2400
agcgctcttc ttagcggggt taacagagct tacccttatg ctgagattgg tgatgagaaa 2460
gtgaaagaac aaatgaacac cttgtttaaa gttctacacc ttgtgaactt cagcaccagc 2520
gtccaggccc tgatgttgct gttccaggtt atggactccc agcagactgt atccgatagg 2580
tactacgcag cactgtataa gaagcttcta gatcccggtt tagcaacctg ctcaaagcca 2640
tccatgtttc ttaatcttgt ctataaatct ctgaaggcag acgtggtgtt acggcgagtg 2700
aaggccttcg tgaagaggtt gcttcaggtc actactggac agacaccacc cttcatttgt 2760
ggaaccctgt accttctgtc tgagcttctg aaagtaaaac caggcctgag ggtccagtta 2820
caggatcacg tggagtctga tgaagaagag tgctttaagg atcaagaaga gactgaagag 2880
aacgaggaaa catttgtgga tgcagacaaa gtagaaaggg aagagaggag cgcaacagaa 2940
aattctgcta aaagaaataa tctgagttca gcagcctcat gggtgcatca tgagaatatg 3000
ggagggagaa agaatggggt ttcctacgat cctttgcacc gaagtccttt atactgtggt 3060
gctgaaagta caagcctttg ggaactgaag aagctttctg aacattttca tccatctgtg 3120
gctctctttg caaaaaccat tctagaggga aatcatattc agtactccgg tgaccctttg 3180
caagatttca cattaatgag attcttggat cgctttgtgt accgaaatcc caaactccac 3240
aaaggcaaag agaacaccag cagtgtggta atgcagcgga agaagaagca gtttatgacg 3300
gatacacaaa atctggcagt caatagtaag gaattccttg ccagagatga aagcaaaatc 3360
ccagtggatg aattattttt tcacagattc tatacaaagt ttgataaaac gcgagagaag 3420
cacaggcgtc aggaggatga agaaagcgtg gaagatgtgg acgatgatga gtttgaaaga 3480
gcactggata catttgaagc tgatggtgct gttggtgttg gccaggatga ccttgatttt 3540
gctggtaata taaaaaagaa aagcaacggg aataagaaaa gcaaaagaag tgaggaatcc 3600
ggtgctgact gggaggattc tggcgatgaa gaattcagtg acctggatga tgaggaagtc 3660
tccttaggaa gcatggagga agatgacttt gaagaggata tggatgagga aggaggtgta 3720
tttatggacg tgtctgatga tggtaatggc ccagacctta acaatgacaa tcaatcgaag 3780
tctgtcagta aaaagaccaa gagaaagaaa gatatgaatt ttatgggatc acatgaagga 3840
tccagccaag gaaagaagag aaaactcaaa gatgccaaaa tactggcagc tgctgaagag 3900
attggctacc tgctggatga aaatgctggg tccaagtttg acaatattgg aatgaatgcc 3960
atggctaaca gagataatgc aaatatcaaa cagatcaagt gggaattaga gcgcgacagg 4020
tggcttcaca acagggatgt gaaaagcatc atcaaaagga aaaaacagct caggcacacg 4080
gggctgaaaa agaagtacag aggcaagaaa tcaaaaagat gaagatctct cgaggtaccg 4140
cggccgcggg gatccagaca tgataagata cattgatgag tttggacaaa ccacaactag 4200
aatgcagtga aaaaaatgct ttatttgtga aatttgtgat gctattgctt tatttgtaac 4260
cattataagc tgcaataaac aagttaacaa caacaattgc attcatttta tgtttcaggt 4320
tcagggggag gtgtgggagg ttttttcgga tcctctagag tcgatctgca ggcatgctag 4380
cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc 4440
acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta 4500
actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca 4560
gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc 4620
cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc 4680
tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat 4740
gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt 4800
ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg 4860
aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc 4920
tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt 4980
ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa 5040
gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta 5100
tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa 5160
caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa 5220
ctacggctac actagaagga cagtatttgg tatctgcgct ctgctgaagc cagttacctt 5280
cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt 5340
ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat 5400
cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat 5460
gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc 5520
aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc 5580
acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta 5640
gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga 5700
cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg 5760
cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc 5820
tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat 5880
cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag 5940
gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat 6000
cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa 6060
ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa 6120
gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga 6180
taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg 6240
gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc 6300
acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg 6360
aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact 6420
cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat 6480
atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt 6540
gccacctgac gtctaagaaa ccattattat catgacatta acctataaaa ataggcgtat 6600
cacgaggccc tttcgtctcg cgcgtttcgg tgatgacggt gaaaacctct gacacatgca 6660
gctcccggag acggtcacag cttgtctgta agcggatgcc gggagcagac aagcccgtca 6720
gggcgcgtca gcgggtgttg gcgggtgtcg gggctggctt aactatgcgg catcagagca 6780
gattgtactg agagtgcacc atatgcggtg tgaaataccg cacagatgcg taaggagaaa 6840
ataccgcatc aggcgccatt cgccattcag gctgcgcaac tgttgggaag ggcgatcggt 6900
gcgggcctct tcgctattac gccagctggc gaaaggggga tgtgctgcaa ggcgattaag 6960
ttgggtaacg ccagggtttt cccagtcacg acgttgtaaa acgacggcca gt 7012
<210> 3
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
ggcccagacc ttaacaatga 20
<210> 4
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
gtcaaacttg gacccagcat 20
Claims (2)
1.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;所述转录因子C/EBPZ的编码基因序列如SEQ ID NO.1所示;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;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;
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;
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。
2.根据权利要求1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PPARγ、FASN、C/EBPα、LPL、FABP4、GATA2或其组合的启动子活性和mRNA表达。
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