L1-ORF2不同片段对报告基因表达产生不同影响  

Different effects on reporter gene expression by distinct L1-ORF2 segements

作　　者：段肖翠[1] 靳霞[1] 谢英[1] 焦宁[2] 刘静[1] 王晓燕[1] 吕占军[1] 

机构地区：[1]河北医科大学实验动物学部遗传研究室,河北省实验动物重点实验室,石家庄050017 [2]河北医科大学第三医院制剂室,石家庄050051

出　　处：《遗传》2009年第1期50-56,共7页Hereditas（Beijing)

基　　金：河北省自然科学基金项目(编号：C2008001065)资助

摘　　要：长散布重复序列-1（Line-1，LI）是重要的人类基因组成分，完整的LI有6kb，在基因组中存在的L，多数是不完整序列，有必要研究LI片段对基因表达的调控作用。PCR扩增LI第二读码框（LI-ORF2）不同位置的280bp片段，共7段，同向8串联按正、反方向分别插入pEGFP质粒GFP基因下游，观察插入序列对GFP报告基因表达的影响。构建的质粒瞬时转染HeLa细胞，经荧光显微镜和Northern检测，不同片段对转录量和终止影响不同。7个片段正序对GFP报告基因的抑制均高于其反序，在正序串联表达载体p280—1＊8和p280—9＊8的GFP基因转录量超过其他280正序插入片段，在反序串联表达载体p280—1＊8as和p280．9＊8as的G即基因转录量超过其他280反序片段。280．1。8、280—9＊8、280—1＊8as和280—9＊8as属于转录终止性序列。Alu在基因组的多数区段与L，分布呈反比，Alu正、反序均对GFP表达有抑制作用，但反序抑制作用高于正序，Alu正序属于转录延伸性序列。280bp片段反序插入的所有质粒荧光阳性细胞均高于正序插入质粒。经碱基分析，L1-ORF2各段均存在A碱基含量多，T碱基含量少的现象，这可能是其正、反序对基因表达影响不同的原因。An intact L1 define element is 6 kb in length in human genome. The majority of the Lls is truncated and has direction difference, implying that it is interesting to study the effects of different length and directions of LIs on gene. In this work, 7 different segments were obtained from LI-open reading frame 2 （ORF2）, each of which was 280 bp in length. Each segment was connected into 8 repeats in head and tail tandem manner and was inserted to downstream of GFP gene in different directions in pEGFP-C1. The inserted ORF2 segments in the sense orientation caused much stronger inhibition on gene transcription and protein expression than antisense sequences did. Among all segments, the first and ninth 280 bp segments of ORF2 in both orientations induced weaker inhibition on gene transcription than other segments in the same orientations and did not induce transcriptional elongation. The distribution of Alu in most regions of genome was inverse ratio with L1. The inserted Alus in both orientations inhibited GFP gene expression, but the inhibition in antisense orienta- tion was stronger than that in sense orientation and the sense Alu was the sequence of inducing transcription elongation. A-rich of ORF2 was probably the molecular basis of its sense orientation with stronger inhibition on gene expression.

关 键 词：L1 ALU PEGFP-C1 ORF2 GFP 

分 类 号：Q75[生物学—分子生物学]