秀丽线虫中p53/cep-1基因克隆、表达及其对寿命的影响

Cloning,Expressing of Caenorhabditis elegans p53/cep-1 and the Effect on Lifespan

Caenorhabditis elegans中的cep-l基因与哺乳动物中的肿瘤抑制因子p53同源,能够引发由DNA损伤导致的细胞凋亡。cep-l基因与寿命的关系研究并不多。我们利用C.elegans的cDNA进行PCR扩增得到cep-l的片段并构建到表达载体以及RNA干扰载体上,经过IPTG诱导表达得到了约90kDa的CEP-1融合蛋白。同时对C.elegans三种虫株:野生型,daf-2(e1370),daf-16(e1038)进行RNAi(RNA-mediated interference),发现cep-l RNAi之后野生型的寿命显著延长,另外两种虫株的寿命没有显著改变,并以RT-PCR的方法检测了cep-l在这三种虫株中的表达情况。

Caenorhabditis elegans cep-1 was similar to its mammalian counterpart, tumor suppressor p53. cep-1 was amplified from C.elegans cDNA by PCR and cloned into expression vector and RNA interference vector. The full length of cep-1 cDNA was 2106bp, containing a 5＇untranslated region of 27 bp and a 3＇untranslated region of 144 bp. The open reading frame was 1935bp which predicted encoding a 645 amino acids protein. CEP-1 recombinant protein was obtained by IPTG induced in Escherichia coli. At the same time, we investigated the effect ofp53/cep-1 on C.elegans lifespan in wild type, daf-2 （e1370）, daf-16 （e1038） strains by RNAi （RNA-mediated interference）, and found that the lifespan of wild type was prolonged significantly in cep-1 RNAi worm, in contrast, the lifespan of the other strains have no difference with the RNAi control worms. And the transcription level of cep- 1 in N2, daf-2 （e1370） and daf-16 （e1038） was determined by RT-PCR.