猪肥胖基因片断的克隆及不同日龄猪肥胖基因表达的差异  

Cloning of Obese (ob) Gene of Swine and the Difference of ob Gene Expression at Different Ages

作　　者：汪以真[1] 初晓娜[1] 黄海青[1] 韩菲菲[1] 

机构地区：[1]浙江大学饲料科学研究所,杭州310029

出　　处：《农业生物技术学报》2005年第2期186-190,共5页Journal of Agricultural Biotechnology

基　　金：国家重点基础研究发展计划项目(973)(No.2004CB117500)资助

摘　　要：从杜长嘉(Duroc×Landrace×Taihu)猪的皮下脂肪中提取基因组RNA,用RT-PCR扩增肥胖基因(obesegene;ob),获得1条504bp的片断,以pGEM-TEasyVector为载体,将该基因片断克隆到大肠杆菌(Escherichiacoli)中。从筛选到的阳性克隆中分离出肥胖基因,测定其序列,分析表明该片段为肥胖基因cDNA的部分序列,编码167个氨基酸组成的多肽,是成熟肽的主体部分。研究得到的基因片段与报道的猪脂肪组织中obcDNA部分序列同源性达到99.41%,氨基酸序列同源性达到98.94%。以ob基因片段的克隆为基础,构建了优化的半定量RT-PCR法,以β-actin为内标,研究不同日龄的猪脂肪组织ob基因表达的差异,1￣28日龄ob基因表达呈上升趋势,28日龄达到最高,随后,到56日龄ob基因表达又呈下降趋势。Genome RNA was extracted from adipose of pig (Duroc×Landrace×Taihu) and obese (ob) gene mRNA was amplified using RT-PCR. A DNA fragment about 504 bp in length was obtained and the PCR product was cloned into pGEM-T vector. The ob gene was isolated and sequenced from the positive clones screened. Sequence analysis suggested that this fragment was partial sequence of ob cDNA and coded 167 amino acid residues, which constituted the major part of leptin mature protein. The gene homology of fragment obtained in this study compared with that of reported ob cDNA sequence in adipocytes of porcine was 99.41%, and amino acid homology is 98.94%. Based on the ob gene clone, an optimal semi-quantitative RT-PCR method was successfully constructed. Using β-actin as inner control, the difference of ob gene expression at different ages of swine was researched, which was increased in suckling stages, and the level rising to a peak by 28 days of age and then declining.

关 键 词：基因表达 不同日龄 基因片断 RT-PCR法 猪 序列同源性 Vector 基因cDNA 肥胖基因 脂肪组织 基因片段 PCR扩增 氨基酸组成 皮下脂肪 Easy 大肠杆菌 阳性克隆 主体部分 上升趋势 RNA 基因组 分析表 成熟肽 半定量 载体 多肽 

分 类 号：Q78[生物学—分子生物学] S892.5[农业科学—特种经济动物饲养]