摘要： 旨在克隆山羊Kruppel样转录因子9（Kruppel-like factor 9，KLF9），阐明其在山羊各组织及其脂肪细胞中的表达模式，为深入探究KLF9基因对山羊肌内脂肪（Intramuscular fat，IMF）沉积的调控作用奠定理论基础。利用胶原酶消化法获得7日龄简州大耳羊羔羊肌内前体脂肪细胞。选成年简州大耳羊与藏山羊各4只，用RT-PCR法克隆山羊KLF9基因，用实时荧光定量PCR方法检测KLF9基因在山羊背最长肌、股二头肌、臂三头肌、皮下脂肪、心脏、肝脏、脾脏、肺脏、肾脏组织中的相对表达水平及KLF9基因在成脂诱导分化0，2，4，6 d的前脂肪细胞中的表达水平。结果显示，获得山羊KLF9基因全长891 bp，其中包括CDS区735 bp，5'UTR序列86 bp，3'UTR序列70 bp，编码244个氨基酸。山羊KLF9氨基酸序列与藏山羊相似性为99.73%，与牛、猪、人、鼠的相似性高达99.18%~97.54%。组织表达结果显示，简州大耳羊肝脏和皮下脂肪的KLF9基因相对表达量极显著高于其他各个组织（P <0.01）；藏山羊肺脏的KLF9基因表达水平极显著高于其他各个组织（P <0.01）。细胞表达检测发现，成脂诱导2 d的KLF9基因前脂肪细胞表达水平极显著高于诱导分化前（P<0.01）。在获得山羊KLF9基因序列基础上，发现了其组织表达模式具有品种特异性，推测其可能在山羊前体脂肪细胞分化早期发挥调控作用。

关键词: 山羊, KLF9, 克隆, 组织表达, 肌内前体脂肪细胞

Abstract: The aim of this study was to clone the sequence of Kruppel-like factor 9 (KLF9) gene and elucidate its expression in different tissues and cells in goats,which may lay a foundation for further study of the KLF9 gene in regulating goat intramuscular fat deposition.Two 7-day-old goats were used for intramuscular preadipocytes culture by collagenase digestion.Various tissues,including heart,liver,spleen,kidney,longissimus dorsi muscle,biceps femoris muscle,triceps brachii muscle and subcutaneous fat,were collected from 4 adult Jianzhou Big-eared goats and Tibetan goats respectively.After total RNA extraction,liver,spleen and subcutaneous fat tissues were used for KLF9 gene cloning by RT-PCR.Real-time fluorescence quantitative PCR (qPCR) method was then used to detect the expression level of KLF9 gene in different tissues and intramuscular pre-adipocytes of different differentiation stages(0,2,4,6 d).The results showed that the length of goat KLF9 gene was 891 bp,including 735 bp CDS,86 bp 5'UTR sequence and 70 bp 3'UTR sequence,encoding 244 amino acids.The KLF9 amino acid sequences of Jianzhou Big-eared goats shared 99.73% similarity with Tibetan goats, following by cattle,pig,human and rat with 99.18%-97.54%,respectively.The expression level of KLF9 gene in liver and subcutaneous fat of Jianzhou Big-eared goats was significantly higher than that in other tissues (P <0.01),while the expression level of KLF9 gene in lungs of Tibetan goats was significantly higher than that in any other tissues(P <0.01).Comparing with the non-differentiation pre-adipocytes,the expression of KLF9 gene was significantly increased in 2 d(P <0.01).The expression pattern of the gene was specific in varieties on the basis of obtaining the KLF9 gene sequence of goat,and it might play an important role in the early stage of differentiation.

Key words: Goat, KLF9, Cloning, Tissue expression, Intramuscular preadipocytes

中图分类号: 

• Q78

• S826