慢肌肌钙蛋白C (Troponin C type 1,TNNC1)具有高度保守性,调控骨骼肌慢肌和心肌的收缩,影响肌蛋白的生成,从而可能导致动物肌肉的生长、进化和功能的差异.本研究以大熊猫和亚洲黑熊骨骼肌为材料,提取总RNA和基因组DNA,运用RT-PCR和Touch-down PCR分别扩增出TNNC1基因的cDNA序列和结构基因序列,并且构建了含有TNNC1 cDNA的重组表达载体,转化进入E.coli BL21进行超表达研究.结果表明大熊猫TNNC1基因的cDNA片段长602 bp,包含一个编码161个氨基酸的开放阅读框,其结构基因全长2 831 bp,包含6个外显子和5个内含子.亚洲黑熊TNNC1基因的cDNA片段长486 bp,亦包含一个编码161个氨基酸的开放阅读框,其结构基因全长2 758 bp,同样包含6个外显子和5个内含子.该两个物种的TNNC1基因与已报道的13种动物的TNNC1基因具有很高的相似性.拓扑预测表明,大熊猫和亚洲黑熊TNNC1蛋白有1个蛋白激酶C磷酸化位点,5个酪蛋白激酶Ⅱ磷酸化位点,1个N-豆蔻酰化位点,3个EF手性钙结合域及1个N-糖基化位点.将TNNC1基因在大肠杆菌中表达发现TNNC1蛋白与氮端多聚组氨酸标签蛋白(His6)融合成大小为23.5kD左右的多肽,这与预期结果相一致.本研究结果为进一步深入探讨大熊猫和亚洲黑熊TNNC1基因及蛋白的结构、功能和进化关系提供资料.%Troponin C type 1 (TNNCl) regulates the contraction of slow-twitch skeletal muscle and cardiac muscle, which may lead to differences in animal muscle growth, evolution, and function. In this study, the genomic sequences and cDNA of the TNNC1 gene of the giant panda (Ailuropoda melanoleuca) and the black bear ( Ursus thibetanus) were cloned successfully using RT-PCR. The TNNCl gene length of cDNA fragment of the giant panda contains an open reading frame of 486 bp encoding 161 amino acids and the length of the genomic sequence is 2 831 bp, contains six exons and five introns. Cloned TNNCl gene cDNA fragment of the black bear contains a 486 bp open reading frame encoding 161 amino acids, the gene structure is 2 758 bp, also contains six exons and five introns. Alignment analysis indicated that the nucleotide sequences and the deduced amino acid sequences for TNNCl gene of the two species are highly conserved with other 13 species which have been reported. Topology prediction shows that there are 5 Casein kinase II phosphorylation sites, 1 Protein kinase C phospho-rylation site, 1 N-myristoylation site, 3 EF-hand calcium-binding domains and 1 N-glycosylation site in the TNNCl protein of the giant panda and the black bear. The cDNA of TNNCl for the giant panda and the black bear were transfected into E. coli, the TNNCl fused with the N-terminally His-tagged protein gave rise to the accumulation of an expected 23. 5 kDa polypeptide, which was consistent with the predicted protein. The results of this study provide information to further explore the structure, function and evolution of TNNCl gene and protein of the giant panda and the black bear.
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