【摘要】 为了研究红鳍东方鲀SREBP-1和SREBP-2基因的功能及在脂肪代谢中的分子机制,从红鳍东方鲀脂肪组织提取总RNA,反转录获得c DNA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c DNA序列长度为3 330 bp,编码1 109个氨基酸,SREBP-2基因ORF区的c DNA序列为3 444 bp,编码1 147个氨基酸。实时荧光定量PCR检测SREBP-1基因在红鳍东方鲀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p ET32a上,利用IPTG对重组质粒p ET32a-SREBP-1在大肠杆菌Rosetta(DE3)进行诱导表达,SDS-PAGE电泳显示在141 k Da处有特异性的条带出现。更多还原

【Abstract】 In order to study the function and molecular mechanism of SREBP-1 and SREBP-2,the open reading frames( ORF) of torafugu SREBP-1 and SREBP-2 genes were amplified from total RNA of adipose tissue by reverse transcription-polymerase chain reaction( RT-PCR). The results showed that the ORF of SREBP-1 was composed of 3 330 bp,encoding 1 109 amino acids,while the ORF of SREBP-2 was composed of 3 444 bp,encoding1 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 p ET32a-SREBP-1 was constructed successfully. SDS-PAGE analysis showed that the expressed protein accumulated and the molecular weight of expressed fusion protein was 141 k Da.更多还原