中文摘要：

目的测定Wistar大鼠c-ski基因部分序列,并应用于检测大鼠c-ski基因表达的实时荧光定量PCR的建立。方法提取培养的Wistar大鼠皮肤成纤维细胞总RNA,通过RT-PCR扩增,将扩增产物克隆到PMD-18T载体上并测序。根据获得的c-ski基因部分序列设计实时荧光定量PCR引物,建立SYBRgreen定量PCR检测方法,并检测培养的大鼠皮肤成纤维细胞c-skimRNA表达水平。结果获得的Wistar大鼠c-ski基因cDNA序列长561bp,为未曾报道的新序列,其与人、小鼠具有较高的同源性,Gen-Bank收录（收入号DQ409171）。建立的SYBRgreen定量PCR检测标准品在10^3～10^9拷贝范围内的相关系数为0.99149。体外培养的Wistar大鼠皮肤成纤维细胞样品中c-skimRNA水平为（1.02±0.18）×10^6拷贝/μl。结论新获得了Wistar大鼠c-ski基因部分序列,该序列可用于实时荧光定量PCR的建立。

英文摘要：

Objective To clone and analyze the partial c-ski gene of Wistar rat,and to develop SYBR Green-based real-time fluorescence quantitative PCR.Methods Total RNA was extracted from the cultured skin fibroblasts of Wistar rat,the partial c-ski gene was then amplified by reverse transcription polymerase chain reaction（RT-PCR） and cloned into eukaryotic expression vector PMD-18 T,then sequenced with Beckman CEQTM8000.The new partial sequence was used to design the oligodoxynucleotide primers,and the real-time quantitative fluorescence PCR was based on SYBR Green,which was obtained to examine the expression of c-ski mRNA in samples of the cultured skin fibroblasts of Wistar rat.Results The partial c-ski gene of Wistar rat obtained by RT-PCR and cloned into eukaryotic expression vector PMD-18 T was 561 bp in length,and shared high homology with that of human and mouse,and accepted by GenBank as new sequence（ACCESSION DQ409171）.The established real-time PCR showed high correlations（r=0.99149） with the standard curves from 10%3 to 10^9 copies.Conclusion The SYBR Green based real-time fluorescence PCR has been established successfully and can be used for determination of c-ski mRNA of skin tissue and cultured cells.