内蒙古白绒山羊蛋白激酶B/AKT基因的克隆及表达模式分析  

Cloning and Expression Pattern of Protein Kinase B/AKT Gene in Inner Mongolia Cashmere Goat

作　　者：杨娇馥[1] 史偈君[1] 梁燕[1] 郑旭[1] 张涛[1] 秦毅[1] 王志钢[1] 刘东军[1] 

机构地区：[1]内蒙古大学生命科学学院/哺乳动物生殖生物学与生物技术教育部重点实验室,呼和浩特010021

出　　处：《中国农业科学》2011年第13期2787-2795,共9页Scientia Agricultura Sinica

基　　金：国家自然科学基金(30860191);转基因生物新品种培育重大专项课题研究计划(2008ZX08008-002);国家基础科学人才培养基金(J0730648)

摘　　要：【目的】克隆内蒙古白绒山羊AKT基因cDNA并分析其基本表达模式。【方法】RT-PCR克隆AKT基因cDNA。通过在线软件BLAST进行核酸序列分析,用SMART与Psite进行氨基酸序列分析。半定量RT-PCR检测AKT基因在绒山羊组织中的表达特异性。Western blotting检测绒山羊胎儿成纤维细胞中AKT表达。【结果】克隆到的内蒙古白绒山羊AKT基因cDNA片段长1 443 bp,包含了编码480个氨基酸残基的全长ORF,氨基酸序列与绵羊(NM_001161857.1)同源性为97%。SMART分析表明,ORF编码的蛋白包含了可与3-磷酸肌醇结合的PH结构域及具有丝氨酸/苏氨酸激酶催化活性的S_TKc结构域。Psite分析表明,含有1个cAMP-/cGMP-依赖性蛋白激酶磷酸化位点、6个蛋白激酶C磷酸化位点、10个酪蛋白激酶Ⅱ磷酸化位点、2个蛋白激酶ATP结合区信号和1个丝氨酸/苏氨酸蛋白激酶活性区域。PSORT程序预测其定位于细胞质中。AKT基因mRNA丰度在睾丸、脑和肾中较高,在脾、肝、肺及乳腺组织中相对低。绒山羊胎儿成纤维细胞中抑制mTOR活性,AKT表达量降低。【结论】内蒙古白绒山羊AKT基因cDNA全长ORF的核苷酸序列与绵羊的AKT基因具有很高的同源性,AKT基因在脾、睾丸、脑、肝、肺、乳腺及肾组织中均有表达,其AKT的表达受mTOR信号通路的调控。[Objective] The present study aims at cloning the CDS fragment ofAKT 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 sofiwares 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 ofAKT 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 ofAKT 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.

关 键 词：内蒙古白绒山羊 AKT 基因克隆 表达模式分析 

分 类 号：S827[农业科学—畜牧学]