绵羊骨骼肌快速肌钙蛋白(TnCf)电子克隆及RT-PCR验证  

In Silico Cloning of Sheep TnCf Gene and Confirmed By RT-PCR

作　　者：徐秋良[1] 张庆莉[1] 唐光武[1] 陈玉林[2] 

机构地区：[1]郑州牧业工程高等专科学校,河南郑州450011 [2]西北农林科技大学动物科技学院,陕西杨凌712100

出　　处：《郑州牧业工程高等专科学校学报》2010年第1期1-4,共4页Journal of Zhengzhou College of Animal Husbandry Engineering

摘　　要：传统的基因克隆有两种方法:一是建立包含目的基因的cDNA文库,根据基因的保守序列设计DNA探针,采用低严谨度的杂交获得目的基因;再者是根据基因的保守序列设计兼并引物,应用PCR技术获得目的基因,这两种方法在操作上有一定的技术难度。我们应用生物信息技术采用电子克隆的方法获得绵羊骨骼肌快速肌钙蛋白基因(TnCf)全编码序列,并用RT-PCR进行验证,结果表明:在TnCf开放阅读框483个碱基中,电子克隆与RT-PCR结果有4个碱基差异,说明电子克隆是基因克隆可靠的生物信息学辅助方法。Identification of sheep fast skeletal muscle troponin C （TnCf） gene based on its sequence conservation has two major approaches, low--stringency hybridization of eDNA libraries, and PCR method using degenerate primers. Both of the two approaches are technically difficult. In current study we have developed an in silico method to isolate the sheep TnCf gene and confirmed the in silico cloning by RT--PCR. After the completely sequencing of cloned eD- NA, we found the sheep TnCf eDNA contained an open reading frame （ORF） consisting of 483 base pairs （bp） that encoded 160 amino acids （aa）. No difference was observed in nucleotide composition of TnCf eDNA between in silieo and cloned eDNA sequences, except for 4 base pairs. It suggested that in silico is an efficient method for gone cloning.

关 键 词：电子克隆 表达序列标签(ESTs) 绵羊骨骼肌快速肌钙蛋白(TnCf) 

分 类 号：S826.99[农业科学—畜牧学]