内蒙古白绒山羊TSC2基因克隆与表达模式分析

Cloning and Expression Pattern of TSC2 Gene in Inner Mongolia Cashmere Goat

旨在克隆内蒙古白绒山羊TSC2基因cDNA并分析其特性及基本表达模式。利用RT-PCR分段克隆TSC2基因cDNA片段并测序,将得到的cDNA各片段核苷酸序列拼接后获得绒山羊TSC2基因编码区全长序列(HQ684023)并进行生物信息学分析。半定量RT-PCR方法检测TSC2基因在不同组织中的表达特异性。结果表明内蒙古白绒山羊TSC2基因cDNA编码区核苷酸序列为5 184 bp,包含了编码1 727个氨基酸残基的全长ORF。核苷酸序列与牛、猪、马、大熊猫、犬、恒河猴、人、小鼠及大鼠的同源性分别为97%、90%、89%、88%、87%、87%、87%、86%和86%。NCBI CDD程序预测该基因编码的蛋白质有一个Tuberin结构域和一个Rap-GAP结构域;Psite程序分析有5个N糖基化位点、2个cAMP和cGMP依赖蛋白激酶磷酸化位点、16个蛋白激酶C磷酸化位点、25个酪蛋白激酶磷酸化位点。PSORT程序预测其定位于胞内体膜。TSC2基因在内蒙古白绒山羊的睾丸、脑、肝脏、肺、乳腺、脾和肾脏等组织中都有表达,mRNA丰度在睾丸中较高,乳腺中较低。

The present study aims at cloning the TSC2 gene cDNA in Inner Mongolia cashmere goat and analyzing its tissue-specific expression pattern.TSC2 gene was cloned by RT-PCR in segmentations.Then,fragments were concatenated by contig method to obtain the full length of the gene.The nucleotide sequence was analyzed with bioinformatics.The tissue-specific expression pattern of TSC2 gene was analyzed by semi-quantitative RT-PCR.The results showed that the cloned TSC2 gene cDNA was 5184 bp in length,including a complete ORF encoding 1727 amino acids.The nucleotide sequence shares 97%,90%,89%,88%,87%,87%,87%,86% and 86% identity with Bos taurus,Sus scrofa,Equus caballus,Ailuropoda melanoleuca,Canis familiari,Macaca mulatta,Homo sapiens,Mus nusculus and Rattus norvegicu.Analysis by NCBI CDD suggested that the encoded protein contained a Tuberin domain and a Rap-GAP domain.Analysis with Psite indicated 5 N-glycosylation site,2 cAMP-/cGMP-dependent protein kinase phosphorylation site,16 Protein kinase C phosphorylation sites and 25 Casein kinase II phosphorylation sites in this protein.Analysis by PSORT suggested that this protein most probably localized in endomembrane.This TSC2 gene was expressed in testicle,brain,liver,lung,mammary glands,spleen and kidney.