中文摘要：

钙调蛋白（calmodulin,CaM）是高度保守的钙离子结合蛋白质,可形成Ca2＋-CaM复合体,从而调节细胞代谢以及靶酶的功能。日本七鳃鳗（Lampetra japonica）作为原始的无颌类脊椎动物,对研究脊椎动物分子起源进化及器官发育分化具有重要的研究价值。通过提取日本七鳃鳗髓组织总RNA,利用RT-PCR方法获得日本七鳃鳗CaM（简称Lj-CaM）基因并进行生物信息学分析。将LjCaM基因分别构建到原核表达载体p ColdⅠ和真核表达载体p EGFP-N1中,利用亲和层析技术纯化得到Lj-CaM蛋白。圆二色谱分析结果表明,Lj-CaM属于典型的α-螺旋结构型蛋白质。免疫印迹和免疫组化结果表明,CaM主要存在于日本七鳃鳗的肠、鳃、髓、肾组织中,在心和肝组织中几乎不表达。细胞免疫荧光结果显示,CaM定位于细胞核中。q PCR和免疫印迹方法检测发现,当293T细胞中Lj-CaM过表达时,对下游靶基因CaMKⅡ作用不明显,但促进PLA2G2A表达。本研究报道了日本七鳃鳗CaM结构、细胞组织定位分布以及基因调控研究,对其结构、分子起源与进化、分子调控及功能方面的研究奠定了基础。

英文摘要：

Calmodulin（ CaM） is a highly conserved calcium-binding protein and able to form Ca2 ＋-CaM complex,thus regulating cell metabolism and function of target enzyme. As an original jawless vertebrate,Lampetra japonica is of great value in the molecular study of origin and evolution of organ differentiation of vertebrates. In this study,total RNA was extracted from the tissue of supraneural body,and the gene of CaM（ Lj-CaM） was obtained by RT-PCR and analyzed by bioinformatics. The Lj-CaM gene was constructed into prokaryotic expression vector p ColdⅠ and eukaryotic expression vector p EGFPN1,respectively. The CaM was purified and analyzed by circular dichroism. The results showed that CaM belonged to typical α helix structure proteins. Western blotting and immunohistochemistry results showed that CaM was mainly expressed in the intestine,gill,supraneural body and kidney. However,expression of CaM was barely detected in heart and liver tissues. Cellular immunofluorescence showedthat CaM was localized in the nucleus. The results of RT-qPCR and Western blotting showed that when the Lj-CaM was overexpressed in 293 T cells,which had no obvious effect on the downstream target protein CaMKⅡ,the expression of PLA2G2 A was up-regulated. In this paper,the structure,distribution and regulation of CaM from lamprey were reported for the first time,providing further understanding of the molecular evolution and function of CaM.