论文部分内容阅读
为了研究红鳍东方 SREBP-1和 SREBP-2基因的功能及在脂肪代谢中的分子机制，从红鳍东方脂肪组织提取总 RNA，反转录获得 cDNA 模板，利用设计的引物 PCR 扩增获得 SREBP-1和 SREBP-2开放阅读框（ORF）， SREBP-1基因 ORF 区的 cDNA 序列长度为3330 bp，编码1109个氨基酸，SREBP-2基因 ORF 区的 cDNA 序列为3444 bp，编码1147个氨基酸。实时荧光定量 PCR 检测 SREBP-1基因在红鳍东方不同组织中的表达特征，结果表明， SREBP-1在脑组织中表达丰度最高，其次为眼、脂肪组织、肠、鳃、脾脏、心脏及肝脏，在肌肉中表达丰度最低。将SREBP-1连接到原核表达载体 pET32a 上，利用 IPTG 对重组质粒 pET32a-SREBP-1在大肠杆菌 Rosetta（DE3）进行诱导表达，SDS-PAGE 电泳显示在141 kDa 处有特异性的条带出现。“,”In order to study the function and molecular mechanism of SREBP-1 and SREBP-2,the open read-ing frames (ORF)of torafugu SREBP-1 and SREBP-2 genes were amplified from total RNA of adipose tissue by re-verse transcription-polymerase chain reaction (RT-PCR).The results showed that the ORF of SREBP-1 was com-posed of 3 330 bp,encoding 1 109 amino acids,while the ORF of SREBP-2 was composed of 3 444 bp,encoding 1 147 amino acids.The expression of SREBP-1 gene in torafugu tissues was determined using Real-time quantitative PCR,presenting that the highest expression was in brain,followed in eye,adipose tissue,spleen,gill,intestine,heart and liver,but the lowest in muscle.The prokaryotic expression system of recombined vector pET32a-SREBP-1 was constructed successfully.SDS-PAGE analysis showed that the expressed protein accumulated and the molecular weight of expressed fusion protein was 141 kDa.