猪TAK1基因的cDNA全长克隆、时空表达及亚细胞定位分析  

Molecular Cloning,Temporal and Spatial Expression Patterns,and Subcellular Localization of Porcine TAK1 Gene

作　　者：李瑞[1] 徐海霞[1,2] 吴伟 王素莹 任梦可 张朋朋 徐永杰 LI Rui;XU Haixia;WU Wei;WANG Suying;REN Mengke;ZHANG Pengpeng;XU Yongjie(College of Life Sciences,Xinyang Normal University,Xinyang 464000,China;Institute for Conservation and Utilization of Agro-bioresources in Dabie Mountains, Xinyang Normal University,Xinyang 464000,China)

机构地区：[1]信阳师范学院生命科学学院,河南信阳464000 [2]信阳师范学院大别山农业生物资源保护与利用研究院,河南信阳464000

出　　处：《华北农学报》2018年第5期68-75,共8页Acta Agriculturae Boreali-Sinica

基　　金：NSFC-河南人才培养联合基金(U1204326);河南省青年骨干教师资助计划项目(2015GGJS-139);河南省教育厅重点科研计划项目(17A230008);信阳师范学院南湖学者计划(2016)

摘　　要：为了研究转化生长因子β激活激酶1(Transforming growth factor-β-activated kinase 1,TAK1)在猪骨骼肌生长发育中的作用,通过cDNA末端快速扩增技术(RACE)获得猪TAK1的cDNA全长,并分析其时空表达及真核细胞中的亚细胞定位情况。结果表明,TAK1 cDNA全长2 163 bp(Gen Bank登录号:KU504629),ORF为1 740 bp,编码579个氨基酸,其氨基酸序列与人、恒河猴、绵羊的相似性均为98. 8%,表明该蛋白在不同物种间高度保守。猪TAK1蛋白具有催化丝氨酸/酪氨酸磷酸化的STKc_TAK1结构域,而在该结构域中还有多个保守的ATP结合位点、A-Loop结构域、TAB1(TGF-beta activated kinase 1)结合位点。荧光定量PCR结果显示,TAK1基因在出生后120 d大白猪的免疫器官脾脏中表达量最高,胰腺、背最长肌等组织中的表达量较低;而不同发育时期的背最长肌中,该基因在胚胎期65 d表达量最高,随着生长发育表达量下调;在不同品种间,TAK1基因在各时期大白猪中的表达量均高于梅山猪,除出生后90 d外,其他时期差异均达到显著水平或极显著水平。pcDNA3. 1(+)-EGFP-TAK1重组质粒转染C2C12细胞后的荧光共定位结果显示,TAK1蛋白的表达主要集中在细胞质中。综上,TAK1基因在猪骨骼肌发育过程中起着重要作用,为进一步研究猪TAK1基因的功能奠定了基础。To investigate the biological role of transforming growth factor-β-activated kinase 1(TAK1)in porcine skeletal muscle development,we cloned the porcine TAK1 cDNA sequences using rapid amplification of cDNA ends(RACE)method.We also detected the temporal and spatial expression patterns and subcellular localization of porcine TAK1 gene.The full-length cDNA sequence of TAK1 was 2 163 bp(GenBank No.KU504629),including a 1 740 bp open reading frame(ORF)that encoded a putative 579 amino acid,and the high sequence similarity of TAK1 between the pig and human,or monkey and or sheep were 98.8%.TAK1 contained a conserved serine/threonine protein kinase catalytic domain(STKc_TAK1),an activation loop(A-Loop),some ATP banding sites and TAB1(TGF-beta activated kinase 1)banding sites.The highly conserved domains indicated that TAK1 had a similar function.The tissue analysis showed that the TAK1 mRNA expression was the highest in pig spleen tissue and very low in pancreas and longissimus dorsi muscle tissue.The result of different developmental stages expression analysis indicated that TAK1 mRNA had the highest expression in embryonic and down-regulation during longissimus dorsi muscle development.Comparing the different breeds,the mRNA abundance of TAK1 in Large White was higher than in Meishan pigs at all development stages,whereas at 90 days,there was no significant difference(P>0.05).Moreover,the fluorescence co-localization showed that the expression of TAK1 was observed mainly in the cytoplasm.These results suggested that TAK1 was probably playing a key role in porcine skeletal muscle development,and made a foundation for the further study on the function of TAK1 in pigs.

关 键 词：猪 TAK1 骨骼肌 克隆 表达 亚细胞定位 

分 类 号：S828[农业科学—畜牧学]