中文摘要：

目的：研究NOR1基因全长及第2外显子选择性剪切异构体在人细胞系和组织中的分布规律以及NOR1蛋白全长及剪切异构体的亚细胞定位模式。方法：以人胎脑cDNA文库质粒为模板,PCR扩增NOR1基因开放阅读框（open reading frame,ORF）,酶切后连接pCMV/myc载体。以pCMV/myc-NOR1（isoform 1）质粒为标准品,采用Real-time RT-PCR方法检测NOR1 mRNA在人胚胎组织和成人睾丸组织中的表达。用RT-PCR方法扩增NOR1基因第2外显子两侧序列片段,测序分析NOR1 isoform 1和isoform 2 mRNA在细胞和组织中的分布;PCR方法扩增细胞系NOR1第2外显子基因组序列,测序检测第2外显子gDNA序列。将pCMV/myc-NOR1（isoform 1）和pCMV/myc-NOR1（isoform 2）质粒转染细胞,采用免疫荧光方法观察两种异构体在细胞内定位模式。结果：从人胎脑cDNA文库克隆得到NOR1基因全长ORF（isoform 1）和遗漏第2外显子的剪切异构体（isoform 2）。Real-time RT-PCR检测发现成人睾丸组织NOR1 mRNA表达水平最高,胚胎鼻咽、气管、脑、肾组织有中等水平表达,其他组织表达较低。RT-PCR结合测序检测发现表明睾丸、鼻咽、气管、肾等16种组织仅表达isoform 2;而脑组织同时检测到NOR1 isoform 1和isoform 2 mRNA,以isoform 2为主。NP69,HNE1等细胞系仅表达遗漏第2外显子的isoform 2,所有检测的细胞系基因组DNA无第2外显子缺失。免疫荧光检测发现NOR1 isoform 1和isoform 2编码蛋白在细胞内定位模式相似。结论：克隆得到NOR1基因全长和遗漏第2外显子的剪切本。Isoform 2是NOR1基因在组织和细胞系中的主要表达形式,NOR1 iso-form 1仅表达于脑组织。第2外显子对NOR1蛋白的细胞内定位模式没有影响。

英文摘要：

Objective To analyze the distribution and subcellular localization of NOR1 alternative splice isoforms in human tissues and cell lines.Methods NOR1 open reading frame（ORF）was amplified from human fetal brain cDNA library and subcloned into pCMV/myc vector.The level of NOR1 mRNA in human tissues was determined by real-time RT-PCR.Region spanning exon 2 was amplified from cDNA or genomic DNA by specific primers and sequenced.The expression plas-mids of NOR1 were transfected into cells and immunofluoresence assay was performed to determine the subcellular localization of NOR1 protein isoforms in human cells.Results The expression of NOR1 mRNA was high in human adult testis,moderate in human fetus nasopharynx,trachea,brain,and kidney tissues,and weak or undetectable in other tissues.Two splice variants of human NOR1 gene resulted from alternative splicing at exon 2 were identified.Both of isoform 1 and isoform 2 mRNA were detected in human fetus brain.Isoform 2 was the sole isoform in other tissues but brain.Only isoform 2 mRNA was detected in cell lines used in this study,though no genomic dele-tion of exon 2 could be found in all these cell lines.Immunofluoresence assay showed both isoform 1 and isoform 2 proteins were distributed in cytoplasm.Conclusion Alternative splice isoforms of tumor suppressor gene NOR1 are identified.NOR1 isoform 1 and isoform 2 are both detected in fetus brain.NOR1 isoform 2 lacking of exon 2 is the sole isoform in multiple tissues except for brain.The exon 2 encoded peptide does not affect the subcellular location of NOR1 protein.