RHD基因第7内含子全长序列分析

目的 通过对"亚洲型"DEL基因第7内含子全长序列测序分析,探讨其Mrna含有来自第7内含子170 bp片段的分子机制.方法 根据美国国家生物信息中心(NCBI)GenBank BN000065的RHD基因序列,设计4对特异性寡核苷酸引物,分四段分别扩增RHD基因第7内含子.测序分析1名正常Rh阳性个体和2名RhDel表型个体(携带RHD1227A等位基因)的RHD第7内含子全长序列,然后通过NCBI Basic BLAST与参考序列(GenBank BN000065)进行比对,并作相互比对.结果 发现3名个体第7内含子共观察到33处碱基变异(GenBank EU372940～2),其中8处变异相同;另有2处碱基变异仅在DEL样本中检出,在正常Rh阳性个体中未发现.结论 这些变异不足以解释以往发现的DEL Mrna存在170 bp来自第7内含子序列而正常D阳性个体却不存在的现象,但测序结果 提示该片段二侧的AG-GT可能参与这一分子事件的形成,因其正好与第7内含子二侧的GT-AG拼接位点协同将内含子一分为二,但具体机制可能十分复杂.

Complete sequencing of RHD intron

Objective To investigate the molecular mechanisms of DEL mRNA with one segment of 170 base pairs from intron 7,the complete nueleotide sequences of Asia DEL intron 7 was analyzed.Methods Four pairs of specific oIigonucleotide primer were designed to amplify and analyze the sequence of RHD intron 7 of one RHD-positive and two DEL with RHD(1227A)allele individuals separately in four sections,according to the RHD sequence of(NCBI)GenBank BN000065.The sequences obtained were aligned each other with a reference of GenBank BN000065 sequence by NCBI Basic BLAST.Results The results showed that 33 nucleotide variants were detected(EMBL/GenBank/DDBJ EU372940～2).Eight variants were observed in all samples whereas two variants were only found in DEL samples,but none in the normal D-positive among them.Conclusions The specific variants in DEL intron 7 cannot demonstrate that DEL mRNA has one segment of 170 base pairs sequence from intron 7,but it is not in normal D-positive individuals.The sequencing results,however,indicate that the AG-GT at both ends of the segment may be concerned with the molecular even.The AG-GT may cut intron 7 into two introns with its normal splicing site(GT-AG),but the molecular mechanisms may be complicate.