干扰山羊KLF12促进皮下脂肪细胞分化  

Knockdown Goat KLF12 to Promote Subcutaneous Adipocytes Differentiation

作　　者：杜宇 王永[1] 孟庆勇[3] 朱江江[1] 林亚秋[1,2] DU Yu;WANG Yong;MENG QingYong;ZHU JiangJiang;LIN YaQiu(Key Laboratory of Qinghai-Tibetan Plateau Animal Genetic Resource Reservation and Utilization,Ministry of Education/Sichuan Province,Southwest Minzu University,Chengdu 610041;College of Animal Science and Veterinary Medicine,Southwest Minzu University,Chengdu 610041;State Key Laboratory for Agrobiotechnology,College of Biological Sciences,China Agricultural University,Beijing 100193)

机构地区：[1]西南民族大学青藏高原动物遗传资源保护与利用教育部/四川省重点实验室,成都610041 [2]西南民族大学畜牧兽医学院,成都610041 [3]中国农业大学生物学院农业生物技术国家重点实验室,北京100193

出　　处：《中国农业科学》2022年第1期184-196,共13页Scientia Agricultura Sinica

基　　金：国家自然科学基金(32072723,31672395);四川省应用基础研究计划重点项目(2018JY0036);西南民族大学中央高校基本科研业务费专项资金(2020PTJS15004)。

摘　　要：【背景】脂肪组织分为皮肤下的皮下脂肪组织(subcutaneous adipose tissue,SAT)和腹部内器官周围的内脏脂肪组织(visceral adipose tissue,VAT),皮下脂肪作为影响肉类美味与否的重要因素,探究皮下脂肪沉积分子调控机制对于育种改良和畜牧业的发展至关重要。Krüppel-like factors 12 (KLF12)是一个进化保守的转录因子,可以在多种细胞类型中表达并控制着广泛的细胞过程。【目的】研究获得山羊KLF12的分子特征并进行生物信息学分析,同时明确KLF12在山羊组织和细胞中的表达模式以及干扰KLF12对山羊皮下脂肪细胞分化的调控作用,为进一步研究KLF12在脂肪沉积过程中的潜在作用提供理论依据。【方法】利用逆转录PCR(reverse transcription PCR,RT-PCR)方法克隆山羊KLF12完整编码序列(coding sequence,CDS)区,使用在线生物信息学分析软件对山羊KLF12核苷酸序列和氨基酸序列进行分析。利用实时荧光定量PCR(Quantitative Real-time PCR,qRT-PCR)技术检测KLF12在山羊心脏、肝脏、腹部脂肪、皮下脂肪、臂三头肌、背最长肌等14个组织中的表达水平,以及诱导分化不同时间段KLF12在皮下前体脂肪细胞中的表达水平。随后,试验通过化学合成山羊KLF12小干扰RNA(si-KLF12),使用Lipofectamine RNAiMAX转染试剂将山羊si-KLF12序列转染到体外培养的山羊皮下前体脂肪细胞中。使用100μmol·L-1油酸导液诱导脂肪细胞分化。利用油红O以及Bodipy染色方法和qRT-PCR技术分别从形态学以及分子生物学的角度阐明干扰KLF12对皮下前体脂肪细胞脂滴积聚和脂肪分化标志基因mRNA表达水平的影响。【结果】试验成功获得包含开放阅读框(open reading frame,ORF)(1 209 bp)的山羊KLF12(1 315 bp,编码402个氨基酸)。亚细胞定位结果显示KLF12主要位于细胞核,此外,KLF12无跨膜结构域和信号肽,且在317—341、347—371及377—399氨基酸处存在3个典型的锌指结构域(ZnF;2H2)【Background】Subcutaneous adipose tissue(SAT) under the skin is an important factor affecting the taste of meat.Exploring the molecular regulation mechanisms of SAT deposition is very important for breeding improvement and the development of animal husbandry. Krüppel-like factors 12(KLF12) is a conserved transcription factor that evolutionarily conserved, and it was found that it could be expressed in a variety of cell types and control a wide range of cellular processes. 【Objective】This study aimed to obtain the coding sequence(CDS) of goat KLF12 and to explore its molecular characteristics. Moreover, the study also intended to clarify the expression pattern of KLF12 in goat tissues and subcutaneous adipocytes, and to explore the role of KLF12 in goat subcutaneous preadipocytes differentiation via interference KLF12, so as to provide a theoretical basis for further research on the potential role of KLF12 in the process of fat deposition. 【Method】In this study, the goat KLF12 CDS sequence was cloned by Reverse Transcription PCR( RT-PCR) method, and the nucleotide sequence and amino acid sequence of goat KLF12 were analyzed on online bioinformatics analysis software. The Quantitative Real-time PCR(q RT-PCR) technology was used to detect the expression levels of KLF12 in goat heart, liver, abdominal fat, subcutaneous fat, triceps brachii, longissimus dorsi and other 14 tissues. Furthermore, the expression level of KLF12 in subcutaneous preadipocytes in different differentiation periods was investigated. Then, the goat KLF12 small interfering RNA(si-KLF12) was chemically synthesized and transfected into goat subcutaneous preadipocyte in vitro by using Lipofectamine RNAiMAX transfection reagent. Subsequently, 100 μmol·L-1 oleic acid induced adipocyte differentiation. Oil red O and Bodipy staining methods and qRT-PCR techniques were used to clarify the effects of interference KLF12 on the accumulation of lipid droplets in subcutaneous preadipocytes and the mRNA expression levels of adipose differentiatio

关 键 词：山羊 KLF12 分子特征 干扰 皮下脂肪细胞 

分 类 号：S827[农业科学—畜牧学]