鸡组织细胞端粒相关序列的克隆及序列分析

Cloning and sequencing of Telomere-associated Sequence in Chicken Tissues

根据真核细胞端粒DNA序列的共同特点 ,设计了一条引物 (TELO0 2 ) ( 2 4nt)。按照TaKala克隆试剂盒的介绍方法 ,染色体基因组DNA经 HaeⅢ 和 HinfⅠ 两种限制性内切酶酶切后 ,先进行初级PCR(C1和TELO0 2 )扩增 ,得到的产物为模板再经过次级PCR(C2和TELO2 )扩增 ,得到的产物用试剂盒回收 ,并连接到PMD_1 8_T载体上 ,转化到JM1 0 9受体菌中。从阳性克隆中提取质粒DNA进行PCR和酶切鉴定 ,确认后进行序列测定 ,获得了一段 2 43sbp的鸡端粒相关序列。此序列与人第 7条染色体基因组末端序列的同源性为 63 .5 % ;与鼠端粒旁序列同源性为 63 .2 % ;而与MDV基因组序列的BamHⅠ酶切片段 76.6%同源。

PCR primer(TELO02)for amplifying telomere associated sequence was designed for telomere repeat sequence in eukaryotes.After genosome DNA of chromosome were digested with HaeⅢ and HinfⅠ .Two PCR had been done as TAKARA LA Clone Kit described,the second PCR products was cloned into pMD18-T Vector.Recombinants were selected and identified by restriction enzyme digestion and PCR.Nucleotide sequence of 243bp 5'-regulatory sequence was determined.Result showed that the 240bp of telomere associated sequence is 63.5%,63.2%,76.6% similary with telomere associated sequence of Human chromosome Ⅶ,Mousr and BamHⅠ fragment of MDV.