【摘要】 旨在克隆山羊KLF8基因序列，阐明组织表达特性，并初步揭示过表达KLF8后对山羊肌内前体脂肪细胞增殖的潜在影响.利用逆转录PCR(reverse transcription-polymerase chain reaction, RT-PCR)方法克隆获得山羊KLF8基因启动子和CDS区序列(coding sequence,CDS),并运用生物信息学方法分析其序列特征；利用基因重组方法构建山羊pcDNA3.1-KLF8真核过表达载体，利用CCK-8、EdU检测方法和实时荧光定量PCR(real-time quantitative PCR,qPCR)明确KLF8对山羊肌内前体脂肪细胞增殖的影响.结果显示，获得包含2个潜在的核心启动子、多个TATA-Box和GATA-Box的山羊KLF8基因启动子2 001bp和包含KLF家族典型的锌指结构域、发挥转录抑制/激活基序的CDS序列1 062 bp.KLF8高表达于山羊脾脏组织，极显著高于其他组织(P<0.01),中度表达于肾脏和肺脏组织，低表达于背最长肌和股二头肌.过表达KLF8抑制山羊肌内前体脂肪细胞增殖，并且这种作用可能是通过上调CCND1的表达来实现的.结果为明确山羊KLF8基因生物学特征及对山羊肌内前体脂肪细胞增殖的影响提供重要的数据支撑.更多还原

【Abstract】 This experiment aimed to clone goat KLF8 gene sequence, elucidate its tissue expression characteristics, and reveal the potential effect of KLF8 on goat intramuscular preadipocytes proliferation through overexpression technique.The promoter and CDS region sequence of goat KLF8 gene was cloned by RT-PCR technique, along with bioinformatics analysis of sequence.pcDNA3.1-KLF8 overexpression vector was constructed by gene recombination method.CCK-8,EdU and real-time quantitative PCR(qPCR) were used to clarify the expression pattern of KLF8 in the proliferation of goat intramuscular preadipocytes and its effect on proliferation.The results showed that the obtained goat KLF8 gene promoter sequence was 2 001bp in length, containing two potential core promoters, multiple TATA-boxes and GATA-boxes, and 1 062 bp of CDS sequence containing typical zinc-finger domain of KLF family and transcriptional repressor/activation motifs.Tissue expression profile assay found that KLF8 was expressed at a high level in spleen tissue and significantly higher than any other tissues(P <0.01),at a medium level in kidney and lung tissue, at a low level in longissimus dorsi muscle and biceps femoris muscle.Overexpression of goat KLF8 inhibited the proliferation of intramuscular preadipocytes, and this regulation may be achieved mainly through up-regulating the expression of CCND1.The results provided important data support to clarify the biological characteristics of goat KLF8 gene and its effect mechanism on the proliferation of goat intramuscular preadipocytes.更多还原