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
罗亚林,硕士生,研究方向:输血相关免疫,E-mail: 812728336@qq.com
纸质出版日期:2021-01-20,
收稿日期:2020-09-04,
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罗亚林,魏玲,姬艳丽等.基于二代测序的RhD阳性个体RHD基因mRNA选择性剪接分析[J].中山大学学报(医学科学版),2021,42(01):51-56.
LUO Ya-lin,WEI Ling,JI Yan-li,et al.Alternative Splicing of RHD mRNA by the Next-generation Sequencing in RhD-positive Individuals[J].Journal of Sun Yat-sen University(Medical Sciences),2021,42(01):51-56.
目的
2
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
2
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
2
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
2
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
2
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
2
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
2
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
2
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.
RhD阳性二代测序转录本剪接
RhD-positivenext-generation sequencingtranscriptssplicing
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