大鼠RVLG选择性剪接变异体的鉴定和分析

doi:10.3864/j.issn.0578-1752.2011.07.021

摘要/Abstract

摘要： 【目的】确认大鼠RVLG（rat vasa-like gene）的第7、8两个内含子的部分片段在卵巢中可能存在选择性剪接现象，进一步检测大鼠RVLG的第7、8两个内含子在卵巢和睾丸中的选择性剪接形式。【方法】从雌雄各5只Wistar大鼠的卵巢和睾丸组织中提取总RNA，反转录成cDNA后作为模板，用PCR扩增RVLG起始密码后约620—690 bp的编码区cDNA。分别选卵巢和睾丸各7个独立克隆测序，将测序结果与大鼠RVLG基因组DNA比对，分析是否有选择性剪接。【结果】大鼠卵巢和睾丸组织中各7个RVLG cDNA 编码区5′端620—690 bp的片段测序结果均存在45 bp和15 bp两处选择性剪接。在7个卵巢RVLG cDNA独立克隆中，有5个仅存45 bp而缺少15 bp，两个仅存15 bp而缺少45 bp；7个睾丸RVLG cDNA独立克隆中，4个仅存在45 bp而缺少15 bp，两个既含有15 bp又含有45 bp，一个既没有45 bp也没有15 bp。经与大鼠RVLG基因组DNA序列比对后确认45 bp位于其第7内含子5′端的两个GT之间，15 bp位于第8内含子3′端的两个AG之间，剪掉或保留这两段均符合典型的GT-AG内含子剪接原则，属选择性剪接。【结论】大鼠RVLG的第7、8两个内含子在卵巢和睾丸中均存在多种选择性剪接形式，体内的标准剪接方式应该是第7外显子3′端保留45 bp，第9外显子不保留来自第8内含子的15 bp。

关键词: RVLG , RT-PCR , 选择性剪接

Abstract: 【Objective】The objective of the study is to confirm the two fragments of introns 7 and 8 of rat RVLG (rat vasa-like gene) pre-mRNA might be alternatively spliced in the ovary, and to further reveal the potential alternative splicing isoforms of introns 7 and 8 of RVLG pre-mRNA both in ovary and testis. In this study the existence of alternative splicing in RVLG pre-mRNA will be further confirmed and the potential alternative splicing isoforms of introns 7 and 8 of RVLG pre-mRNA both in ovary and testis will be tested. 【Method】 The cDNA template was prepared by reverse transcription of total RNA extracted from the ovaries of 5 female rats and the testes of 5 male rats. Partial coding region of 620-690 bp including the initiation codon were amplified by PCR. Fourteen independently amplified fragments, 7 each from ovary and testis, were sequenced and aligned with the genomic DNA of RVLG gene to analyze the alternative splicing pattern. 【Result】 In all the 14 sequenced cDNA, two fragments, one 45 bp and another 15 bp, were found to be involved in alternative splicing patterns. Of the 7 from ovary, 5 retained the 45 bp but lost the 15 bp, 2 retained the 15 bp but lost the 45 bp. Four from testes retained the 45 bp but lost the 15 bp, 2 retained both the 45 bp and the 15 bp, and one without the 45 bp and the 15 bp. The 45 bp was identified to locate at the 5’ end of intron 7 flanked by two GT, and the 15 bp at the 3’ end of the intron 8 flanked with two AG, granting these two fragments cryptic splice site for their alternative splicing.【Conclusion】There are multiple combinations of alternative splicing patterns of introns 7 and 8 of RVLG. The in vivo canonical splicing isoform may be the one that exon 8 with the 45 bp from intron 7, but exon 9 without the 15 bp from intron 8.

Key words: RVLG , RT-PCR , alternative splicing

黄文强, 董珍珍, 邢万金. 大鼠RVLG选择性剪接变异体的鉴定和分析[J]. 中国农业科学, 2011, 44(7): 1474-1480.

HUANG Wen-qiang, DONG Zhen-zhen, XING Wan-jin . Identification and Characterization of the Alternative Splicing Variants of Rat RVLG[J]. Scientia Agricultura Sinica, 2011, 44(7): 1474-1480.

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