野生一粒小麦着丝粒RCS1相关序列的克隆鉴定

用水稻着丝粒重复序列RCS1为探针 ,与 30 72个克隆进行菌落杂交 ,得到了 32个阳性克隆 ,用RCS1与拟斯卑尔脱山羊草着丝粒重复序列Tcs2 5 0为探针进一步筛选 ,在 32个RCS1相关的阳性克隆中任选 10个克隆进行点杂交 ,分别有 6个和 5个阳性克隆 .为了克隆RCS1相关片段 ,依据RCS1的序列设计了三对引物 ,将引物 3从上述阳性克隆中扩增的一个 5 4 3bp的片段克隆测序 ,发现与水稻RCS1部分片段达到约 83%的同源 ,与大麦的反转座子 (Ty3 gypsy)部分序列同源性达到了 92 % ,与节节麦中着丝粒的整合酶基因部分序列同源性达到了 96 % ,命名为TBRCS1.TBRCS1可能是野生一粒小麦着丝粒区的组成部分

Isolation and characterization of centromere RCS1 homology sequence from Triticum boeoticum

In order to study the structure and clone the sequence of the centromere of T. boeoticum , 3072 clones of the BAC library were screened by RCS1 as probes. Thirty two positive clones were obtained. Then ten positive clones picked at random from the thirty two positive clones wre probed by RCS1 and Tcs250 respectively again. Six and five clones showed unambiguous positive hybridization respectively. Then three pairs of primers to RCS1 were designed using DNA star software. PCR amplification was carried out using positive BAC clones as templates by the three pairs of primers respectively. We obtained a 600 bp (named TBRCS1) sequence by primer 3. Sequencing analysis revealed that TBRCS1 had 83% homology to RCS1 in rice and 92% to a Ty3/gypsy class of retrotransposon sequence in barley and 96% to a integrase like gene reported in Aegilops tauschii It indicated that TBRCS1 was part of centromere of T. boeoticum.