红鳍东方 SREBP-1和 SREBP-2基因克隆及表达分析

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。

Cloning and Expression of SREBP-1 and SREBP-2 in Torafugu Takifugu rubripes

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.