水蕨中心体蛋白基因的克隆与原核表达

蕨类植物的精子形成是一个运动细胞器重新发生的过程,但对其精子发生的分子机制仍不甚了解。中心体蛋白(centrin)是定位于细胞中心体上的一种高度保守的钙结合蛋白,被认为可能是最早参与运动细胞器发生的蛋白质。克隆鉴定centrin蛋白为进一步分析蕨类精子发生的分子机制奠定基础。该研究从蕨类模式植物水蕨(Ceratopteris thalictroides)中克隆到了水蕨centrin基因(CtCEN),其cDNA序列全长为1 077bp,结构分析表明CtCEN基因属于EF-hand超家族的centrin基因。Centrin的系统树分析发现,藻类、蕨类以及动物等具鞭毛游动精子的生物聚为一支,而被子植物聚为另一分支,表明centrin的功能可能与运动细胞器鞭毛发生有关。原核表达和Western blot分析表明,CtCEN基因表达的蛋白能与人源centrin抗体发生结合,这一结果证实克隆到了centrin基因。

Clone and Prokaryotic Expression of Centrin Gene from the Fern Ceratopteris thalictroides

In ferns,the spermatogenesis is a process of de novo formation of the motile apparatuses and the molecular mechanisms of the spermatogenesis were unclear.Centrin,a highly conservative calcium binding protein usually locating in the centrosome,was considered to be the protein that participates the formation of motile apparatuses.Cloning of the centrin gene has some significance for elucidating the molecular mechanism of spermatogenesis of ferns.The present investigation cloned the centrin gene (CtCEN) from the fern model plant,Ceratopteris thalictroides.The full length the cDNA sequence of the CtCEN gene is 1 077 bp,and the structural analysis showed that the gene belongs to be centrin gene of EF hand protein superfamily.Phylogenetic analysis indicated that the CtCEN of the algae,ferns,animal and human,all of which produce sperms with flagella,is clustered in one branch.The CtCEN of angiosperm is clustered in another branch.It is suggested that the centrin may participate the genesis of flagellum.The prokaryotic expression and Western blot analysis showed that the expressed protein of CtCEN can hybridize with the centrin protein antibody of Human,which indicated that the CtCEN belongs to the centrin family.The present studies lay a foundation for the molecular mechanism of the centrin participating formation of motile apparatuses in fern spermatogenesis.