关键词: 内蒙古白绒山羊, AKT, 基因克隆, 表达模式分析

Abstract:   【Objective】The present study aims at cloning the CDS fragment of AKT gene cDNA in Inner Mongolia Cashmere Goat and analyzing its tissue-specific expression. 【Method】AKT gene cDNA was cloned by RT-PCR. The nucleotide sequence was analyzed by BLAST and amino acid sequence was analyzed by online softwares SMART and Psite. The tissue-specific expression pattern of AKT was analyzed by semi-quantitative RT-PCR. The expression of AKT in goat fibroblasts was detected by Western blotting.【Result】The cloned AKT gene cDNA was 1 443 bp in length, including a complete ORF encoding 480 amino acids. The amino acid sequence shares 97% identity with the sheep AKT (NM_001161857.1). Analysis by SMART suggested that the encoded protein contained a PH domain which can bind 3-phosphoinositides and an S_TKc domain which possess serine/threonine kinase catalytic activity. Analysis with Psite indicated one cAMP-/cGMP-dependent protein kinase phosphorylation site, 6 protein kinase C phosphorylation sites, 10 casein kinase II phosphorylation sites, 2 protein kinases ATP-binding region signatures and one serine/threonine protein kinases active-site signature in this protein. Analysis by Psort suggested that this protein most probably localizes in cytoplasm. The expression of AKT mRNA was higher in testicle, brain and kidney, while lower in spleen, liver, lung and mammary glands. The expression of AKT in goat fibroblasts was inhibited when the cells were treated by mTOR specific inhibitor CCI-779 for 48 hours. 【Conclusion】The full length of AKT gene ORF in Inner Mongolia Cashmere Goat was cloned and shared high identity with sheep. AKT gene was shown to express in testicle, brain, kidney, spleen, liver, lung and mammary glands. The expression of AKT in goat fibroblasts was regulated by mTOR signaling pathway.

Key words: Inner Mongolia Cashmere Goat, AKT, Gene cloning, Expression pattern