基于二代测序的RhD阳性个体RHD基因mRNA选择性剪接分析  

Alternative Splicing of RHD mRNA by the Next-generation Sequencing in RhD-positive Individuals

作　　者：罗亚林 魏玲[2] 姬艳丽[2] 罗广平[2] 付涌水 温机智[2] 黎诚耀[1] LUO Ya-lin;WEI Ling;JI Yan-li;LUO Guang-ping;FU Yong-shui;WEN Ji-zhi;LI Cheng-yao(Department of Transfusion Medicine,Southern Medical University,Guangzhou 510515,China;Institute of Clinical Blood Transfusion,Guangzhou Blood Center,Guangzhou,510095,China)

机构地区：[1]南方医科大学检验与生物技术学院输血医学系,广东广州510515 [2]广州血液中心临床输血研究所,广东广州510095

出　　处：《中山大学学报（医学科学版）》2021年第1期51-56,共6页Journal of Sun Yat-Sen University:Medical Sciences

基　　金：国家自然科学基金(81900184)。

摘　　要：【目的】本研究旨在对RhD阳性个体中红细胞特异性表达的RHD基因不同转录本进行分析,并对选择性剪接发生的机制进行初步探讨。【方法】利用体外有核红细胞培养体系分离并培养RhD阳性个体的有核红细胞,使用逆转录聚合酶链反应(RT-PCR)技术对RhD阳性个体RHD基因外显子6至3’非编码区扩增后进行二代测序,分析mRNA转录本,并采用生物信息学方法分析RHD基因所有外显子5’及3’剪接位点的最大熵值。【结果】RHD基因的转录本以正常全长mRNA为主,除此之外还存在其他8种异常转录本。按照表达量从多到少,分别为外显子7缺失、外显子9缺失、外显子8和9缺失、外显子7、8和9缺失、外显子7和9缺失、外显子8和9缺失合并外显子7与10间插入一段170 bp的内含子7片段、全长mRNA但插入170 bp的内含子7片段和外显子9缺失但插入一170 bp的内含子7片段,其中最后3种为新转录本。生物信息学分析提示外显子7和外显子9异常剪接可能与其5’-剪接位点(5’-splice site,5’ss)或3’ss和剪接体结合能力下降有关,而内含子7片段异常表达于转录本中可能与其两端存在类似5’ss和3’ss剪接保守序列有关。【结论】RHD基因存在十分复杂的选择性剪接模式,使RhD阳性个体的RHD基因在mRNA水平上存在多种不同的剪接转录本。【Objective】This study aimed to analyze different transcripts specifically expressed by red blood cells in RhD-positive individuals,and to explore the mechanism of alternative splicing.【Methods】Erythroblasts from RhD-positive individuals were isolated and cultured in an in vitro erythroblast culture system.The fragment from exon 6 to 3’untranslated coding regions of RHD gene of RhD-positive individuals was amplified by reverse transcription polymerase chain reaction(RT-PCR)method.The PCR products were then sequenced by next-generation sequencing and the mRNA transcripts were analyzed.The maximum entropy values of 5’splice site(ss)and 3’ss of all exons of RHD gene were analyzed using bioinformatics.【Results】The transcripts of the RHD gene were mainly normal full-length mRNA.In addition,eight other abnormal transcripts were also identified.According to the expression frequencies(high to low),these transcripts were exon 7 deletion,exon 9 deletion,exons 8 and 9 deletion,exons 7-9 deletion,exons 7 and 9 deletion,exons 8-9 deletion and 170 bp insertion between exon 7 and 10,full-length with 170 bp insertion between exon 7 and 8,exon 9 deletion and 170 bp insertion between exon 7 and 8.The last three transcripts were novel.Bioinformatic analysis suggested that the abnormal splicing of exon 7 and exon 9 might be due to the decreased binding ability of 5’ss or 3’ss to the spliceosome.The intron 7 was expressed in mRNA,which might be related to the sequences conserved with 5’ss and 3’ss splicing.【Conclusion】The alternative splicing patterns of RHD gene are very complicated,which leads to various transcripts at the mRNA level of the RHD gene from RhD-positive individuals.

关 键 词：RhD阳性 二代测序 转录本 剪接 

分 类 号：R331.1[医药卫生—人体生理学] R41[医药卫生—基础医学]