猪FGL2基因cDNA末端序列检测及结构分析

Detection of cDNA End Sequence of Porcine Fibrinogen Like Protein2 and It′s Structure Analysis

检测猪FGL2基因cDNA末端序列并对该基因结构初步分析。α 32PdCTP放射性同位素标记cDNA探针筛选猪基因组DNA文库;cDNA末端快速扩增(rapidamplificationofcDNAend,RACE)。以猪正常小肠及心脏组织提取新鲜总RNA,反转录后作为模板,设计基因特异性引物,采用Advantage2聚合酶混合物进行PCR扩增;依据猪与人FGL2基因3′端已知同源序列设计PCR上游引物,以人FGL2基因3′末端序列设计下游引物,以猪基因组DNA为模板采用Advantage2聚合酶混合物进行PCR反应;PCR载体重组质粒DNA亚克隆扩增。同位素探针未能筛选到特异阳性克隆,RACE反应检测到特异性转录起始位置及第一个转录终止位置,但仍未检测到第二个转录终止位置。猪基因组DNA行PCR扩增成功检测到猪FGL2基因3′末端未知序列及第二个转录终止位置。

The purpose of this study is to detect the end sequence of porcine FGL2 gene cDNA and make a preliminary analysis of it′s structure.Porcine DNA library was screened by a cDNA probe labeled with radioactive isotope α32P dCTP.Rapid amplification of cDNA end (RACE):Retroverse transcription product of total RNA extracted from normal porcine tissue was used as the template,gene specific primers were designed and advantage 2 polymerase mix was used in PCR,of which using porcine genomic DNA as the template:forward primer was designed according to the acquired consensus region of human and pig FGL2 3′ sequences while reverse primer was designed from human FGL2 3′ end downstream sequence;TA cloning.Screening library failed to get any specific positive clone;the specific transcription initiation site and first poly A signal were successfully detected by RACE reaction although it fails again to detect the second poly A signal.The unknown sequence of porcine FGL2 3′ end including the second poly A signal was successfully detected by PCR using genomic DNA as the template.RACE reaction can be applied as an effective method to detect the specific transcription initiation and termination sites.Using advantage 2 polymerase mix instead of regular DNA polymerase may significantly improve the sensitivity,accuracy and specificity of PCR reaction.If it met particular difficulties in the regular screening of DNA library,PCR reaction utilizing primers designed from the known consensus sequence and genomic DNA as template may be considered as an appropriate alternative.