大鼠FMR1同源基因cDNA的克隆、测序与选择剪接的初步分析

Cloning and Sequencing of the cDNA of the rat FMR1 Gene and Primary Analysis of its Alternative Splicing

应用ＲＴ－ＰＣＲ方法，从新生大鼠脑组织总ＲＮＡ扩增大鼠ＦＭＲ１同源基因的ｃＤＮＡ片段，克降至ｐＵＣ１８质粒中进行序列分析．获得从终止密码子起共１６８１ｂｐ的编码序列，尚缺少约２００ｂｐ的５′序列．所克隆的这部分大鼠ＦＭＲ１ｃＤＮＡ，不含有对应于人ＦＭＲ１基因的外显子１２及外显子１７第一和第三剪接受点之间的序列，提示大鼠ＦＭＲ１基因也有选择剪接表达．同源性分析显示，大鼠ＦＭＲ１与小鼠ＦＭＲ１基因的同源性为９７．７％，与人ＦＭＲ１基因的同源性为９４．９％；与小鼠ＦＭＲＰ（ＦＭＲ１蛋白）的氨基酸序列同源性为９８．４％，与人ＦＭＲＰ的氨基酸序列同源性为９７．９％．以大鼠ＦＭＲ１ｃＤＮＡ片段为探针检测到大鼠不同组织中ＦＭＲ１基因的选择剪接表达．上述结果为以大鼠为动物模型深入研究ＦＭＲ１基因功能奠定了基础．

The abnormal expression of FMR1 gene is considered the molecular basis of fragile X syndrome.The cDNA sequences of human and mouse have been reported.The partial cDNA sequence of FMR1 homologous gene was analyzed in rat.With RT PCR,three overlapped fragments covering most part of FMR1 coding area were amplified from neonatal rat brain and then cloned and sequenced.The rat sequence showed alternative exclusion of exon 12 and partial exon 17 suggested the existence of splicing events in rat.While the rat FMR1 gene exhibits marked sequence identity with mouse as 97 7% and with human as 94 9%,comparison of the amino acid sequences of rat with mouse and rat with human FMR1 also revealed strong homology,with similarity values as 98 4% and 97 9%.Furthermore,with rat cDNA fragment as probe,analysis by Southern blot hybridization with RT PCR products demonstrated different transcripts in different tissues of rat.These results facilitate further characterization of FMR1 gene in rat,which would be an appropriate supplement model for human.